Agents useful for reducing amyloid precursor protein and treating dementia and methods of use thereof

ABSTRACT

The present invention provides compounds and methods of administering compounds to a subject that can reduce βAPP production and that is not toxic in a wide range of dosages. The present invention also provides non-carbamate compounds and methods of administering such compounds to a subject that can reduce βAPP production and that is not toxic in a wide range of dosages. It has been discovered that either the racemic or enantiomerically pure non-carbamate compounds can be used to decrease βAPP production.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of and claims the benefit ofU.S. application Ser. No. 11/455,959, filed Jun. 20, 2006 now abandoned,which claims priority to U.S. application Ser. No. 10/415,765, filedFeb. 6, 2004, now issued as U.S. Pat. No. 7,153,882, which is a 35U.S.C. §371 national phase application from, and claims priority to,International Application PCT/US01/48175, filed Nov. 2, 2001 andpublished under PCT Article 21(2) in English, which claims priority toU.S. Provisional Application No. 60/245,329, filed Nov. 2, 2000, whichapplications are incorporated herein in their entireties by thisreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to agents useful for reducing amyloidprecursor protein and methods of use thereof.

2. Background Art

The major pathological hallmarks of Alzheimer's disease (AD), aprogressive neurodegenerative condition leading to loss of memory, arecharacterized by the appearance of senile plaques which are primarilycomposed of Aβ and neurofibrillary tangle aggregates (Selkoe, 1997;Roberson and Harrell, 1997). Aβ, a 40-42 residue peptide, is derivedfrom a larger protein, βAPP (695-770 amino acids), whose biologicalfunctions remain to be fully determined but whose pathological role maybe separated on the basis of its final proteolysed form (Checker, 1995;Selkoe, 1997). βAPP derivatives are generated by three enzymaticactivities termed α-, β- and γ-secretases, to produce different proteinfragments that are either neuroprotective or amyloidogenic. An aspartylprotease with β-secretase like properties has been identified (Hussaainet al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al.,1999), that may serve as a therapeutic marker. However, its value as atarget for drug development is complicated by its location within twomembranes (plasma and Golgi apparatus). Furthermore, the role ofalternative compensatory activities remains unclear. Indeed, a secondenzyme, Thimet oligopeptidase, was found capable of β-secretase activityin transfected COS cells (Koike et al., 1999). A major pharmaceuticalindustry focus has been to look for agents that reduce amyloidogenicprocessing using compounds that can manipulate βAPP to producenon-amyloidogenic by-products. However, it is important to note that therole of alternative βAPP fragments in AD is unclear.

Regarding regulatory mechanisms involved in βAPP processing,environmental agents have been demonstrated to accelerate βAPP turnoverinto its pathological Aβ form (Selkoe, 1997). Furthermore, the cellularsurrounding of neurons, particularly astrocytes and microglia, areadditional and non-neuronal sources of βAPP (Funato et al., 1998;Akiyama et al., 2000). Thus, amyloid plaque occurrence is oftenassociated with enlarged microglia which produce interleuken-1 (IL-1), apotent mediator of astroglial proliferation and βAPP production (Akiyamaet al., 2000). The fact that IL-1 can influence this process suggeststhat signaling pathways induced by cytokines are interconnected withβAPP metabolism Another example of receptor-signaling association andβAPP homeostasis is demonstrated through the activation of muscarinic m1and m3 receptors which modify βAPP synthesis and processing through MAPkinase dependent and independent pathways (Felder et al., 1993; Nitschet al., 1992 and 1994). Reductions in muscarinic receptors, as in AD,may alter βAPP metabolism and result in subsequent Aβ deposition.Cholinergic system impairment has been reversed with moderate success bythe use of anticholinesterases (Greig et al., 1995; Brossi et al.,1996), the only approved drugs for AD treatment.

A family of novel anticholinesterases, phenserine and analogues, hasbeen synthesized. Phenserine dramatically improves cognitive performancein rodents and is in clinical trails (Greig et al., 1995; Patel et al.,1998). Studies of rats with forebrain cholinergic lesions that are knownto dramatically increase βAPP in cholinergic projection areas have shownthat phenserine can protect against this and additionally, reduce βAPPproduction in naive animals (Haroutunian et al., 1997). As both βAPPprocessing and cholinesterase activity are affected in the AD brain(Bronfman et al., 1996) and as the anticholinesterase, tacrine, has beenshown to decrease βAPP and Aβ in neuronal cells in vitro (Lahiri et al.,1998), current studies have focused on the molecular changes induced byphenserine. In these studies, naturally-occurring phenserine (the(−)-enantiomer) was used.

It is the cholinergic action of anticholinesterases such as(−)-phenserine, rivastigmine (Exellon®, Novartis®), donepezil (Aricept®,Pfizer®), galanthamine (Jansen®), tacrine (Cognex®, Warner Lambert®),(−)-physostigmine (Synapton®, Forest®), that provides the compoundstheir ability to improve cognitive performance in both animal models andhumans. Likewise, it is the cholinergic action that is also doselimiting for these same compounds (nausea, sweating, GI effects) (Beckeret al., 1991). Conversely, the (+)-enantiomers are unable to inhibiteither acetylcholinesterase (AChE., EC 3.1.1.7.) orbutyrylcholinesterase (BChE., EC 3.1.1.8.), and hence have nocholinergic action. The (+)-enantiomers are also unnatural isomers andthus, need to be synthesized. Synthetic procedures provide a mixture of(+)- and (−)-forms that require early separation into optically pureforms to eventually obtain the final products.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

SUMMARY OF THE INVENTION

The present invention provides compounds and methods of administeringcompounds to a subject that can reduce βAPP production and that is nottoxic in a wide range of dosages. The present invention also providesnon-carbamate compounds and methods of administering such compounds to asubject that can reduce βAPP production and that is not toxic in a widerange of dosages. It has been discovered that either the racemic orenantiomerically pure non-carbamate compounds can be used to decreaseβAPP production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the synthesis of analogues of (+)-physostigmine.

FIG. 2 shows the transformation of (+)-oxindole to analogues of(+)-physostigmine. Compound numbers refer to compounds in Table 1.

FIG. 3 shows effects of (+) and (−)-phenserine treatment of SH-SY5Yneuroblastoma cells on βAPP protein levels and ERK transcription factorlevels.

FIG. 4 shows effects of (−)-phenserine treatment of SK-N-SHneuroblastoma cells on extracellular βAPP protein levels (4A),intracellular βAPP protein levels (4B), toxicity (4C) and Aβ levels(4D).

FIG. 5 shows Aβ and βAPP levels in SK-N-SH cells after administration of(+)-phenserine.

FIG. 6 shows (−)-phenserine treatment of U373 MG astrocytoma alone or incombination with ERK and PI 3 kinase inhibitors.

FIG. 7 shows βAPP protein levels in SK-N-SH cells after administrationof cymserine and its analogs.

FIG. 8 shows effects of (−)-phenserine treatment on reporter geneexpression in the presence and absence of the βAPP-mRNA 5′ UTR (8A),intracellular βAPP protein levels (8B), and on βAPP RNA levels (8C) intransfected U373 MG astrocytoma cells.

FIG. 9 shows KNAPP levels in transgenic mice after administration of(−)-phenserine and (−)-phenethylcymserine.

FIGS. 10A-10B show Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in transgenic mice afteradministration of (−)-phenserine and (−)-phenethylcymserine derived fromanimals described in FIG. 9.

FIG. 11A shows the translational regulation of phenserine (Rate of APPSynthesis) in SH-SY5Y cells. FIG. 11B shows the effects of(−)-phenserine on steady state APP mRNA levels in SH-SY5Y cells.

FIGS. 12A and B show the effects of (−)-phenserine on extracellular andintracellular APP levels in SK-N-SH, respectively.

FIG. 13A shows the effects of (−)-phenserine on secreted APP levels andcell-viability in SH-SY5Y cells. FIGS. 13B-M show the effects of severalcompounds of the invention on secreted APP levels and cell-viability inSH-SY5Y cells.

FIG. 14 shows the effects of several compounds of the invention onextra- and intracellular APP levels in SH-SY5Y cells.

FIG. 15A shows the translational regulation by compounds (rate of APPSynthesis) in SH-SY5Y cells using several compounds of the invention.FIG. 15B shows the effects of several compounds of the invention on thesteady state APP mRNA levels in SH-SY5Y cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of desired embodiments of the inventionand the Examples included therein.

Before the present compounds, compositions and methods are disclosed anddescribed, it is to be understood that this invention is not limited tospecific synthetic methods, as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

Throughout this application, where publications and patents arereferenced, the disclosures of these publications and patents in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art to which thisinvention pertains.

Variables, such as R₁-R₁₅, n, A, D, E, G, X, Y, and Z throughout theapplication are the same variables as previously defined unless statedto the contrary.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeaning.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon group of 1 to 4, 1 to 8, or 1 to 20 carbon atoms,such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,pentyl, hexyl, heptyl, octyl, and the like. Examples of cycloalkylgroups include cyclopentyl and cyclohexyl.

The term “alkenyl” as used herein refers to a hydrocarbon group of 2 to4, 2 to 8, or 2 to 20 carbon atoms and structural formula containing acarbon-carbon double bond.

The term “alkynyl” as used herein refers to a hydrocarbon group of 2 to4, 2 to 8, or 2 to 20 carbon atoms and a structural formula containing acarbon-carbon triple bond.

The term “aryl” is defined as any carbon-based aromatic group including,but not limited to, phenyl, benzene, naphthalene, anthracene,phenanthrene, pyrene, and benzo[a]pyrene, etc.

The term “substituted aryl” is defined as an aryl group having at leastone group attached to the aryl group that is not hydrogen. Examples ofgroups that can be attached to the aryl group include, but are notlimited to, alkyl, alkynyl, alkenyl, aryl, heterocyclic, halide, nitro,amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, alkoxy, cyano,alkoxy, thioalkyl, haloalkyl, hydroxyalkyl, alkylamino, diakylamino, oracyl. In various embodiments, a substituent is bound to carbon 2, 3, 4,5, or 6 of one of these moieties. Examples of alkoxy substituentsinclude, but are not limited to, methoxy, ethoxy, and isopropoxy groups.Examples of acyl substituents include acetyl and benzoyl groups.

The term “aralkyl” is defined as an aryl group having an alkyl, alkynyl,or alkenyl group attached to the aryl group. An example of an aralkylgroup is a benzyl group.

The term “heteroaryl” is defined as an aryl group that has at least oneheteroatom such as nitrogen, sulfur, or oxygen incorporated within thering of the aryl group.

The term “heteroalkyl” is defined as an alkyl group that has at leastone heteroatom, such as nitrogen, sulfur, oxygen, or phosphate,incorporated within the alkyl group or attached to the alkyl group.

The invention, in one aspect, relates to a compound having the formula Ior II

-   -   wherein R₁ and R₂ are, independently, hydrogen, branched or        straight chain C₁-C₈ alkyl, substituted or unsubstituted aryl,        or aralkyl;    -   R₃ is branched or straight chain C₁-C₄ alkyl or heteroalkyl or        C₄-C₈ alkyl or heteroalkyl, or substituted or unsubstituted        aryl;    -   X and Y are, independently, O, S, alkyl, hydrocarbon moiety,        C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently, hydrogen,        oxygen, branched or straight chain C₁-C₈ alkyl, C₂-C₈ alkenyl or        C₂-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl;        and    -   R₆ is hydrogen; C₁-C₈ alkyl, C₁-C₈ alkenyl, C₂-C₈ alkynyl,        aralkyl, or substituted or unsubstituted aryl, or (CH₂)_(n)R₇,        where R₇ is hydroxy, alkoxy, cyano, ester, carboxylic acid,        substituted or unsubstituted amino, and n is from 1 to 4,    -   wherein the compound having the formula I or II is the        substantially pure (+)-enantiomer,    -   with the proviso that the compound is not (+)-physostigmine,        (+)-octylcarbamoyleseroline, (+)-benzylcarbamoyleseroline,        (+)-physovenine, (+)—N-methylphysostigmine, (+)-phenserine,        (+)-3-(1-methylamino)-ethyl-2H-indol-5-yl-phenylcarbamate,        (+)-N¹-benzylnorphysostigmine, (+)-N′-benzylnorphenserine,        (+)-N¹-benzylnortolserine, (+)-N¹-benzylnorcymserine,        (+)-N¹-norphysostigmine, (+)-N¹-norphenserine,        (+)-N¹-nortolserine, (+)-N′-norcymserine,        (+)—N⁸-benzylnorphysostigmine, (+)—N⁸-benzylnorphenserine,        (+)—N⁸-norphysostigmine, (+)—N⁸-norphenserine,        (+)—N¹,N⁸-bisbenzylnorphysostigmine,        (+)—N¹,N⁸-bisbenzylnorphenserine, (+)—N¹,N⁸-bisnorphysostigmine,        or (+)—N¹,N⁸-bisnorphenserine,    -   with the proviso that when the compound is formula I, X is NCH₃,        and Y is NR₅, where R₅ is hydrogen, loweralkyl, arylloweralkyl,        heteroarylloweralkyl, cycloalkylmethyl, or loweralkenyl methyl,        R₃ is not methyl or at least one of R₁ or R₂ is not H or alkyl.

The invention also relates to a compound having the formula III or IV

-   -   wherein R₁ and R₂ are, independently, hydrogen, branched or        straight chain C₁-C₈ alkyl, substituted or unsubstituted aryl,        or aralkyl;    -   R₃ is branched or straight chain C₁-C₄ alkyl, or substituted or        unsubstituted aryl;    -   X and Y are, independently, O, S, alkyl, hydrocarbon moiety,        C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently, hydrogen,        oxygen, branched or straight chain C₁-C₈ alkyl, q-C₈ alkenyl, or        C₂-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl;        and    -   R₆ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        aralkyl, or substituted or unsubstituted aryl, or (CH₂)_(n)R₇,        where R₇ is hydroxy, alkoxy, cyano, ester, carboxylic acid,        substituted or unsubstituted amino, and n is from 1 to 4,

The chiral center of compounds I-IV is the carbon atom that has R₃bonded to it. Here, the (+)-enantiomer has R₃ pointing behind the planeof the page. In various embodiments, the compounds having the structureI or II have an enantiomeric purity for the (+)-enantiomer of from 55 to100%, desirably from 75 to 100%, more desirably from 85 to 100%, moredesirably from 95 to 100%, and even more desirably 100%.

In one embodiment, when the compound is formula I, R₃ is methyl and X isNCH₃.

In one embodiment, when the compound is formula I or II, R₃ is notmethyl. In particular embodiments, R₃ is a branched or straight chainalkyl or heteroalkyl group of 2, 3, 4, 5, 6, 7, or 8 carbons orsubstituted or unsubstituted aryl.

In another embodiment, when the compound has the structure I or II, Y isC(H)R₄ or X is O, S, or C(H)R₄.

In another embodiment, when the compound is formula I, R₃ is methyl, Xis NCH₃, and Y is NCH₃. In one embodiment, when the compound is formulaI, R₃ is methyl, X is NCH₃, Y is NCH₃, and R₁ is C₁-C₈ straight chainalkyl or benzyl and R₂ is hydrogen. In one embodiment, when the compoundis formula I, R₃ is methyl, X is NCH₃, Y is NCH₃, and R₁ is substitutedor unsubstituted phenyl and R₂ is hydrogen. In one embodiment, when thecompound is formula I, R₃ is methyl, X is NCH₃, Y is NCH₃, and R₁ and R₂are, independently, methyl or ethyl.

In another embodiment, when the compound is formula I, R₃ is methyl, Xis NCH₃, and Y is O. In one embodiment, when the compound is formula I,R₃ is methyl, X is NCH₃, Y is O, R₁ is C₁-C₈ straight chain alkyl orbenzyl, and R₂ is hydrogen. In one embodiment, when the compound isformula I, R₃ is methyl, X is NCH₃, Y is O, and R₁ and R₂ are,independently, methyl or ethyl. In one embodiment, when the compound isformula I, R₃ is methyl, X is NCH₃, Y is O, and R₁ is substituted orunsubstituted phenyl and R₂ is hydrogen.

In another embodiment, when the compound is formula I, R₃ is methyl, Xis NCH₃, and Y is S. In one embodiment, when the compound is formula I,R₃ is methyl, X is NCH₃, Y is S, R₁ is C₁-C₈ straight chain alkyl orbenzyl, and R₂ is hydrogen. In one embodiment, when the compound isformula I, R₃ is methyl, X is NCH₃, Y is S, and R₁ and R₂ are,independently, methyl or ethyl. In one embodiment, when the compound isformula I, R₃ is methyl, X is NCH₃, Y is S, R₁ is substituted orunsubstituted phenyl, and R₂ is hydrogen.

In another embodiment, when the compound is formula I, R₃ is methyl, Xis NCH₃, and Y is NR₅. In one embodiment, when the compound is formulaI, R₃ is methyl, X is NCH₃, and Y is NR₅, wherein R₅ is —CH₂CH═CH₂,—CH₂CH₂Ph, benzyl, or hydrogen.

In another embodiment, when the compound has the formula I, R₃ ismethyl, Y is NCH₃, and X is NCH₃, wherein R₄ is benzyl or hydrogen.

In another embodiment, when the compound is formula I, R₃ is methyl, Xis NCH₃, Y is NR₅, wherein each R₄ and R₅ is, independently, hydrogen orbenzyl.

In another embodiment, when the compound is formula I, R₃ is phenyl, Xis NCH₃, and Y is NCH₃.

In another embodiment, when the compound is formula I, R₃ is methyl, andX is NCH₃, and Y is not NH or NHCH₂Ph.

In another embodiment, when the compound is formula II, R₃ is methyl, Xis C(H)CH₃, and R₆ is (CH₂)₂R₇, where R₇ is a substituted orunsubstituted amino group.

In another embodiment, the compound having the formula I or II can befound in Table 1. Although only the (+)-isomer is illustrated to savespace, it is the intent of the invention to claim the (+)-isomer,(−)-isomer, and mixtures of both isomers (e.g., racemic 1:1 mixtures) ofall of the compounds of the invention unless such compounds arespecifically excluded.

TABLE 1 Structure No. R Compounds

 1  2  3  4  5  6  7  8  9  10 CH₃ C₂H₅ (CH₂)₂CH₃ CH₂(CH₃)₂ (CH₂)₃CH₃(CH₂)₄CH₃ (CH₂)₅CH₃ (CH₂)₆CH₃ (CH₂)₇CH₃ CH₂C₆H₅ (+)-Physostigmine(+)-Ethylcarbamoyleseroline (+)-Propylcarbamoyleseroline(+)-Isopropylcarbamoyleseroline (+)-Butylcarbamoyleseroline(+)-Pentylcarbamoyleseroline (+)-Hexylcarbamoyleseroline(+)-Heptylcarbamoyleseroline (+)-Octylcarbamoyleseroline(+)-Benzylcarbamoyleseroline

 11  12  13  14  15  16  17  18  19  20 CH₃ C₂H₅ (CH₂)₂CH₃ CH₂(CH₃)₂(CH₂)₃CH₃ (CH₂)₄CH₃ (CH₂)₅CH₃ (CH₂)₆CH₃ (CH₂)₇CH₃ CH₂C₆H₅(+)-Physovenine (+)-Ethylcarbamoylphysovenol (+)-Propylcarbamoylphysovenol (+)-Isopropylcarbamoyl physovenol (+)-Butylcarbamoylphysovenol (+)-Pentylcarbamoyl physovenol (+)-Hexylcarbamoyl physovenol(+)-Heptylcarbamoylphysovenol (+)-Octylcarbamoyl physovenol(+)-Benzylcarbamoyl physovenol

 21  22  23  24  25  26  27  28  29  30 CH₃ C₂H₅ (CH₂)₂CH₃ CH₂(CH₃)₂(CH₂)₃CH₃ (CH₂)₄CH₃ (CH₂)₅CH₃ (CH₂)₆CH₃ (CH₂)₇CH₃ CH₂C₆H₅(+)-Thiaphysovenine (+)-Ethylcarbamoylthiaphysovenol(+)-Propylcarbamoylthia physovenol (+)-Isopropylcarbamoylthia physovenol(+)-Butylcarbamoylthia physovenol (+)-Pentylcarbamoylthia physovenol(+)-Hexylcarbamoylthia physovenol (+)-Heptylcarbamoylthiaphysovenol(+)-Octylcarbamoylthia physovenol (+)-Benzylcarbamoylthia physovenol

 31  32 CH₃ C₂H₅ (+)-N-Methylphysostigmine (+)-Diethylcarbamoyleseroline

 33  34 CH₃ C₂H₅ (+)-N-Methylphysovenine (+)-Diethylcarbamoylphysovenol

 35  36 CH₃ C₂H₅ (+)-N-Methylthiaphysovenine(+)-Diethylcarbamoylthiaphysovenol

 37  38  39  40  41  42  43  44  45  46  47  48  49  50  51  52  53  54H 2′-CH₃ 3′-CH₃ 4′-CH₃ 2′-CH₂CH₃ 3′-CH₂CH₃ 4′-CH₂CH₃ 2′-CH₂(CH₃)₂3′-CH₂(CH₃)₂ 4′-CH₂(CH₃)₂ 2′,3′-CH₃ 2′,4′-CH₃ 2′,5′-CH₃ 2′,6′-CH₃3′,4′-CH₃ 3′,5′-CH₃ 3′,6′-CH₃ 2′,4′,6′-CH₃ (+)-Phenserine (+)-Tolserine(+)-3′-Methylphenserine (+)-4′-Methylphenserine (+)-2′-Ethylphenserine(+)-3′-Eethylphenserine (+)-4′-Eethylphenserine(+)-2′-Isopropylphenserine (+)-3′-Isopropylphenserine (+)-Cymserine(+)-2′,3′Dimethylphenserine (+)-2′,4′-Dimethylphenserine(+)-2′,5′-Dimethylphenserine (+)-2′,6′-Dimethylphenserine(+)-3′,4′-Dimethylphenserine (+)-3′,5′-Dimethylphenserine(+)-3′,6′-Dimethylphenserine (+)-2′,4′,6′-Trimethylphenserin

 55  56  57  58  59  60  61  62  63  64  65  66  67  68  69  70  71  72H 2′-CH₃ 3′-CH₃ 4′-CH₃ 2′-CH₂CH₃ 3′-CH₂CH₃ 4′-CH₂CH₃ 2′-CH₂(CH₃)₂3′-CH₂(CH₃)₂ 4′-CH₂(CH₃)₂ 2′,3′-CH₃ 2′,4′-CH₃ 2′,5′-CH₃ 2′,6′-CH₃3′,4′-CH₃ 3′,5′-CH₃ 3′,6′-CH₃ 2′,4′,6′-CH₃(+)-Phenylcarbamoyl-physovenol (+)-2′-Methylphenylcarbamoyl-physovenol(+)-3′-Methylphenylcarbamoyl-physovenol (+)-4′-Methylphenylcarbamoyl-physovenol (+)-2′-Ethylphenylvarbamoyl-physovenol(+)-3′-Eethylphenylcarbamoyl-physovenol(+)-4′-Eethylphenylcarbamoyl-physovenol(+)-2′-Isopropylphenylcarbamoyl-physovenol(+)-3′-Isopropylphenylcarbamoyl-physovenol(+)-4′-Isopropylphenylcarbamoyl-physovenol(+)-2′,3′-Dimethylphenylcarbamoyl-physovenol(+)-2′,4′-Dimethylphenylcarbamoyl-physovenol(+)-2′,5′-Dimethylphenylcarbamoyl-physovenol(+)-2′,6′-Dimethylphenylcarbamoyl-physovenol(+)-3′,4′-Dimethylphenylcarbamoyl-physovenol(+)-3′,5′-Dimethylphenylcarbamoyl-physovenol(+)-3′,6′-Dimethylphenylcarbamoyl-physovenol(+)-2′,4′,6′-Trimethylphenylcarbamoyl-physovenl

 55  56  57  58  59  60  61  62  63  64  65  66  67  68  69  70  71  72H 2′-CH₃ 3′-CH₃ 4′-CH₃ 2′-CH₂CH₃ 3′-CH₂CH₃ 4′-CH₂CH₃ 2′-CH₂(CH₃)₂3′-CH₂(CH₃)₂ 4′-CH₂(CH₃)₂ 2′,3′-CH₃ 2′,4′-CH₃ 2′,5′-CH₃ 2′,6′-CH₃3′,4′-CH₃ 3′,5′-CH₃ 3′,6′-CH₃ 2′,4′,6′-CH₃(+)-Phenylcarbamoyl-thiaphysovenol(+)-2′-Methylphenylcarbamoyl-thiaphysovenol(+)-3′-Methylphenylcarbamoyl-thiaphysovenol (+)-4′-Methylphenylcarbamoyl-thiaphysovenol(+)-2′-Ethylphenylvarbamoyl-thiaphysovenol(+)-3′-Eethylphenylcarbamoyl-thiaphysovenol(+)-4′-Eethylphenylcarbamoyl-thiaphysovenol(+)-2′-Isopropylphenylcarbamoyl-thiaphysovenol(+)-3′-Isopropylphenylcarbamoyl-thiaphysovenol(+)-4′-Isopropylphenylcarbamoyl-thiaphysovenol(+)-2′,3′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-2′,4′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-2′,5′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-2′,6′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-3′,4′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-3′,5′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-3′,6′-Dimethylphenylcarbamoyl-thiaphysovenol(+)-2′,4′,6′-Trimethylphenylcarbamoyl-thiaphysovenol

 73  74  75  76 CH₃ phenyl 2′-tolyl 4′-cymyl(+)-N¹-Allylnorphysostigmine (+)-N¹-Allylnorphenserine(+)-N¹-Allylnortolserine (+)-N¹-Allylnorcymserine

 77  78  79  80 CH₃ phenyl 2′-tolyl 4′-cymyl(+)-N¹-Phenethylnorphysostigmine (+)-N¹-Phenethylnorphenserine(+)-N¹-Phenethylnortolserine (+)-N¹-Phenethylnorcymserine

 81    82    83    84 CH₃   phenyl   2′-tolyl   4′-cymyl(+)-3-(1-Methylamino)-ethyl-2H-indol-5yl- methylcarbamate(+)-3-(1-Methylamino)-ethyl-2H-indol-5yl- phenylcarbamate(+)-3-(1-Methylamino)-ethyl-2H-indol-5yl- tolylcarbamate(+)-3-(1-Methylamino)-ethyl-2H-indol-5yl- cymylcarbamate

 85  86  87  88 CH₃ phenyl 2′-tolyl 4′-cymyl(+)-N¹-Benzylnorphysostigmine (+)-N¹-Benzylnorphenserine(+)-N¹-Benzylnortolserine (+)-N1-Benzylnorcymserine

 89  90  91  92 CH₃ phenyl 2′-tolyl 4′-cymyl (+)-N¹-Norphysostigmine(+)-N¹-Norphenserine (+)-N¹-Nortolserine (+)-N¹-Nororcymserine

 93  94  95  96 CH₃ phenyl 2′-tolyl 4′-cymyl(+)-N⁸-Benzylnorphysostigmine (+)-N⁸-Benzylnorphenserine(+)-N⁸-Benzylnortolserine (+)-N⁸-Benzylnorcymserine

 97  98  99 100 CH₃ phenyl 2′-tolyl 4′-cymyl (+)-N⁸-Norphysostigmine(+)-N⁸-Norphenserine (+)-N⁸-Nortolserine (+)-N⁸-Norcymserine

101 102 103 104 CH₃ phenyl 2′-tolyl 4′-cymyl(+)-N¹,N⁸-Bisbenzylnorphysostigmine (+)-N¹,N⁸-Bisbenzylnorphenserine(+)-N¹,N⁸-Bisbenzylnortolserine (+)-N¹,N⁸-Bisbenzylnorcymserine

105 106 107 108 CH₃ phenyl 2′-tolyl 4′-cymyl(+)-N¹,N⁸-Bisnorphysostigmine (+)-N¹,N⁸-Bisnorphenserine(+)-N¹,N⁸-Bisnortolserine (+)-N¹,N⁸-Bisnorcymserine

109 110 111 112 CH₃ phenyl 2′-tolyl 4′-cymyl (+)-3a-Phenylphysostigmine(+)-3a-Phenylphenserine (+)-3a-Phenyltolserine (+)-3a-Phenylcymserine

The invention, in one aspect, a compound having the formula XIV or XV

-   -   wherein R₁ and R₂ are, independently, hydrogen, branched or        straight chain C₁-C₈ alkyl, substituted or unsubstituted aryl,        or aralkyl;    -   R₃ is branched or straight chain C₁-C₄ alkyl or heteroalkyl or        C₄-C₈ alkyl or heteroalkyl, or substituted or unsubstituted        aryl;    -   X and Y are, independently, O, S, alkyl, hydrocarbon moiety,        C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently, hydrogen,        oxygen, branched or straight chain C₁-C₈ alkyl, C₂-C₈ alkenyl,        or C₂-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl;        and    -   R₆ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        aralkyl, or substituted or unsubstituted aryl, or (CH₂)_(n)R₇,        where R₇ is hydroxy, alkoxy, cyano, ester, carboxylic acid,        substituted or unsubstituted amino, and n is from 1 to 4,    -   wherein the compound having the formula XIV or XV is a racemic        mixture or the substantially pure (−)-enantiomer.

In one embodiment, the compounds having the structure XV and XV have anenantiomeric purity for the (−)-enantiomer of from 55 to 100%, desirablyfrom 75 to 100%, more desirably from 85 to 100%, more desirably from 95to 100%, and even more desirably 100%.

In another embodiment, when the compound has the structure XIV or XV, Yis C(H)R₄ or X is O, S, or C(H)R₄

In yet another embodiment, the compound of formula XIV is MES9280 (FIG.13K), EES9232 (FIG. 13J), MES9313 (FIG. 13F), or a (+)-isomer or aracemic mixture thereof.

In another embodiment, when the compound has the formula XIV, where Xand Y are nitrogen, and the compound is the substantially pure(−)-enantiomer, then R₃ is not methyl. In another embodiment, when thecompound has the formula XIV, where X is nitrogen, Y is nitrogen oroxygen, and the compound is the substantially pure (−)-enantiomer, thenR₅ is not hydrogen or C₁-C₁₀ alkyl. In another embodiment, when thecompound has the formula XIV, where X is nitrogen and Y is sulfur, andthe compound is the substantially pure (−)-enantiomer, then R₃ is notmethyl. In another embodiment, the compound is not (−)-phenserine,(−)-physostigmine, (−)-heptyl-physostigmine, (−)-physovenine,(−)-N(1)-norphysostigmine, MES9217 (FIG. 13H), MES9299 (FIG. 13L), andMES9329 (FIG. 13M). In one embodiment, when the compound is formula XIVor XV, R₃ is not methyl. In particular embodiments, R₃ is a branched orstraight chain alkyl or heteroalkyl group of 2, 3, 4, 5, 6, 7, or 8carbons or substituted or unsubstituted aryl.

FIGS. 1 and 2 show one approach to the synthesis of compounds having thestructure I and II. Using techniques known in the art (see Julian etal., J. Am. Chem. Soc., 1935, 57, 563; Lee et al., J. Org. Chem., 1991,56, 872; Pei et al., Heterocycles, 1995, 41, 2823; Lee et al., J.Chromatography, 1990, 523, 317; and Pei et al., Heterocycles, 1994, 39,557, which are incorporated by reference in their entirety), thephysostigmine compound A (FIG. 1) can be prepared in high yield andenantiopurity. Compound A is an important intermediate, and can be usedto produce a variety of compounds having the structure I and II. FIG. 2shows that by using techniques known in the art (see Yu et al.,Heterocycles, 1988, 27, 745; Yu et al., Helv. Chem. Res., 1991, 74, 761;He et al., Med. Chem. Res., 1992, 2, 229; Pei et al., Med. Chem. Res.,1995, 5, 455; Yu et al., J. Med. Chem., 1998, 31, 2297; Zhu, Tet. Lett.,2000, 41, 4861; Pei et al., Med. Chem Res., 1995, 5, 455; and Yu et al.,J. Med. Chem., 1997, 40, 2895, which are incorporated by reference intheir entirety) compound A can be converted to a number of differentcompounds having the structure I and II. The numbers below each compoundin FIG. 2 correspond to the compound numbers in Table 1. The racemicmixture of I and II as well as compounds XIV and XV can be preparedusing the synthetic procedure outlined in FIGS. 1 and 2, where a chiralchromatography step is not performed to produce the racemic mixture orthe (−)-enantiomer is isolated instead of the (+)-enantiomer.

The invention also relates to a compound having the formula V, VI, orVII

-   -   wherein R₈ is hydrogen, branched or straight chain C₁-C₈ alkyl,        substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where        R₉ and R₁₀ are, independently, hydrogen or alkyl, and R₁₁ is        alkyl;    -   R₃ is branched or straight chain C₁-C₄ alkyl, or substituted or        unsubstituted aryl;    -   X and Y are, independently, O, S, C(H)R₄, or NR₅, wherein R₄ and        R₅ are, independently, hydrogen, oxygen, branched or straight        chain C₁-C₈ alkyl, C₂-C₈ alkenyl, or C₂-C₈ alkynyl, aralkyl, or        substituted or unsubstituted aryl; and    -   R₆ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl,        aralkyl, or substituted or unsubstituted aryl, or (CH₂)_(n)R₇,        where R₇ is hydroxy, alkoxy, cyano, ester, carboxylic acid,        substituted or unsubstituted amino, and n is from 1 to 4,    -   with the proviso that when the compound is formula V, X is NCH₃,        and Y is NR₅, where R₅ is hydrogen, loweralkyl, arylloweralkyl,        heteroarylloweralkyl, cycloalkylmethyl, or loweralkenyl methyl,        R₃ is not methyl or R₈ is not H or, loweralkyl.

The compounds having the formula V-VII as well as compounds VIII-XIIIdescribed below are referred to herein as “non-carbamate compounds”because they do not possess the carbamate group present in compoundsI-IV, XIV-XVI. Compounds V-XIII are generally the synthetic precursorsto compounds I-IV. FIG. 1 provides a general synthesis to intermediateA, which is a species of compound V. Intermediate A is shown as the(+)-enantiomer; however, the (−)-enantiomer can also be isolated usingchiral chromatography. Similarly, the (+)- and (−)-enantiomers ofcompound VI can also be produced using similar methodology. Furthermore,in the absence of the chiral chromatography step in FIG. 1, the racemiccompounds V and VI can be produced.

In another embodiment, when the non-carbamate compound has the formulaVII, where X is NR₅, the reaction depicted in Scheme 1 can be used toproduce the compounds. For example, in Scheme I, compound B (compoundVII where X is NH) can be treated with a base, such as NaNH₂, thentreated with an alkyl or aralkyl halide compound to produce compound C.

In one embodiment, when the compound is formula V, X and Y are NR₅,wherein R₅ is branched or straight chain C₁-C₈ alkyl, desirably methyl.In another embodiment, when the compound is formula V, R₃ is methyl, Xand Y are NCH₃, and R₈ is C₁-C₈ straight chain alkyl, desirably methyl.

In another embodiment, when the compound is formula VII, X is NR₅,wherein R₅ is branched or straight chain C₁-C₈ alkyl or aralkyl,desirably benzyl. In another embodiment, when the compound is formulaVII, R₆ is (CH₂)_(n)R₇, where R₇ is a substituted or unsubstituted aminogroup. In another embodiment, when the compound is formula VII, X isNR₅, where R₅ is benzyl, R₆ is (CH₂)₂N(CH₃)₂, and R₈ is methyl.

The invention also relates to a compound having the formula VIII

wherein R⁸ is hydrogen, branched or straight chain C₁-C₈ alkyl,substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where R₉ andR₁₀ are, independently, hydrogen or alkyl, and R₁₁ is alkyl;R³ is branched or straight chain C₁-C₄ alkyl, or substituted orunsubstituted aryl;X is O, S, C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently,hydrogen, oxygen, branched or straight chain C₁-C₈ alkyl, C₁-C₈ alkenyl,or C₂-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl; andR⁶ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aralkyl, orsubstituted or unsubstituted aryl, or (CH₂)_(n)R₇, where R₇ is hydroxy,alkoxy, cyano, ester, carboxylic acid, substituted or unsubstitutedamino, and n is from 1 to 4.

The invention further relates to a compound having the formula IX

wherein R⁸ is hydrogen, branched or straight chain C₁-C₈ alkyl,substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where R₉ andR₁₀ are, independently, hydrogen or alkyl, and R₁₁ is alkyl;R³ is branched or straight chain C₁-C₄ alkyl, or substituted orunsubstituted aryl;R⁶ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aralkyl, orsubstituted or unsubstituted aryl, or (CH₂)_(n)R₇, where R₇ is hydroxy,alkoxy, cyano, ester, carboxylic acid, substituted or unsubstitutedamino, and n is from 1 to 4;X is O, S, C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently,hydrogen, oxygen, branched or straight chain C₁-C₈ alkyl, C₂-C₈ alkenyl,or C₂-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl; andA, D, and E are, independently, hydrogen, hydroxy, alkoxy, halide,alkyl, aralkyl, or amino.

The invention also relates to a compound having the formula X

wherein R⁸ is hydrogen, branched or straight chain C₁-C₈ alkyl,substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where R₉ andR₁₀ are, independently, hydrogen or alkyl, and R₁₁ is alkyl;R⁶ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aralkyl, orsubstituted or unsubstituted aryl, or (CH₂)_(n)R₇, where R₇ is hydroxy,alkoxy, cyano, ester, carboxylic acid, substituted or unsubstitutedamino, and n is from 1 to 4;X is O, S, C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently,hydrogen, oxygen, branched or straight chain C₁-C₈ alkyl, C₂-C₈ alkenyl,or C₂-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl; andZ is halide, hydroxy, or alkoxy.

The invention also relates to a compound having the formula XI

wherein R⁸ is hydrogen, branched or straight chain C₁-C₈ alkyl,substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where R₉ andR₁₀ are, independently, hydrogen or alkyl, and R₁₁ is alkyl;R⁶ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aralkyl, orsubstituted or unsubstituted aryl, or (CH₂)_(n)R₇, where R₇ is hydroxy,alkoxy, cyano, ester, carboxylic acid, substituted or unsubstitutedamino, and n is from 1 to 4;X is O, S, C(H)R₄, or NR₅, wherein R₄ and R₅ are, independently,hydrogen, oxygen, branched or straight chain C₁-C₈ alkyl, C₂-C₈ alkenyl,or q-C₈ alkynyl, aralkyl, or substituted or unsubstituted aryl;A, D, E, and G are, independently, hydrogen, hydroxy, alkoxy, halide,alkyl, aralkyl, or amino; andZ is halide, hydroxy, or alkoxy.

In one embodiment, steps 3-5 in FIG. 1 can be used to prepare compoundshaving the formula VII-XI.

The invention further relates to a compound having the formula XII

wherein R⁸ is hydrogen, branched or straight chain C₁-C₈ alkyl,substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where R₉ andR₁₀ are, independently, hydrogen or alkyl, and R₁₁ is alkyl;R⁶ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, aralkyl, orsubstituted or unsubstituted aryl, or (CH₂)_(n)R₇, where R₇ is hydroxy,alkoxy, cyano, ester, carboxylic acid, substituted or unsubstitutedamino, and n is from 1 to 4; andR¹² and R¹³ are, independently, hydrogen; C₁-C₈ alkyl; aryl orsubstituted aryl; aralkyl; or (CH₂)_(n)R¹⁴, wherein R¹⁴ is hydroxy,alkoxy, ester, carboxylic acid, substituted or unsubstituted amino, andn is from 1 to 4.

The invention also relates to compound having the formula XIII

wherein R⁸ is hydrogen, branched or straight chain C₁-C₈ alkyl,substituted or unsubstituted aryl, aralkyl, or CR₉R₁₀OR₁₁, where R₉ andR₁₀ are, independently, hydrogen or alkyl, and R₁₁ is alkyl;R⁶ is hydrogen; C₁-C₈ alkyl, C₂-C₈ alkenyl, q-C₈ alkynyl, aralkyl, orsubstituted or unsubstituted aryl, or (CH₂)_(n)R₇, where R₇ is hydroxy,alkoxy, cyano, ester, carboxylic acid, substituted or unsubstitutedamino, and n is from 1 to 4; andR¹² and R¹³ are, independently, hydrogen; C₁-C₈ alkyl; aryl orsubstituted aryl; aralkyl; or (CH₂)_(n)R¹⁴, wherein R¹⁴ is hydroxy,alkoxy, ester, carboxylic acid, substituted or unsubstituted amino, andn is from 1 to 4; andA, D, E, and G are, independently, hydrogen, hydroxy, alkoxy, halide,alkyl, aralkyl, or amino.

In one embodiment, compounds having the formula XII and XIII can beprepared by the general reaction shown in Scheme 2.

The invention also relates to a compound having the formula XVI

-   -   wherein R₁ and R₂ are, independently, hydrogen, branched or        straight chain C₁-C₈ alkyl, substituted or unsubstituted aryl,        or aralkyl;    -   R₃ is branched or straight chain C₁-C₄ alkyl, or substituted or        unsubstituted aryl;    -   R₅ and R₁₅ are, independently, hydrogen, oxygen, branched or        straight chain C₁-C₈ alkyl, C₂-C₈ alkenyl, or C₂-C₈ alkynyl,        aralkyl, or substituted or unsubstituted aryl; and    -   wherein the compound is a racemic mixture, the substantially        pure (−)-enantiomer, or the substantially pure (+)-enantiomer.

In a related aspect, the invention features the compound disclosed inFIG. 13C (MES9287) and MES9286 (Table 2).

Additional non-carbamate compounds of the present invention are shown inTable 2.

TABLE 2 9206

9201

9199

9191

9226

9205

9203

9202

9230

9225

9222

9215

9236

9229

9228

9227

9243

9235

9234

9231

9266

9242

9238

9237

9275

9260

9259

9257

9279

9271

9270

9276

9295

9278

9277

9276

9305

9293

9292

9291

9311

9302

9301

9295

9317

9310

9309

9306

9234

9316

9314

9312

9323

9321

9319

9318

9320

9330

9335

9331

9286

9287

9290

In one embodiment, the present invention provides a method of inhibitingproduction of amyloid precursor protein in a cell, comprising contactingthe cell with a compound having the formula I-XVI and any combinationthereof. As used herein, “inhibiting” means decreasing the amount orconcentration of amyloid precursor protein. “Inhibition” also refers tohalting or reducing the production of amyloid protein precursor, whereinthe concentration of amyloid protein precursor is reduced. Thus, theinhibition of production of amyloid precursor protein can be measured,for example, by comparing the amount of amyloid precursor proteinproduced by cells after contacting the cells with the compound havingthe formula I-XIII and any combination thereof, with the amount ofamyloid precursor protein produced by control cells that have not beencontacted with a compound having the formula I-XIII and any combinationthereof. In one embodiment, the cell that is contacted with the compoundis in vivo, ex vivo, or in vitro. The cell of this invention can be amammalian cell, desirably a human cell.

In a desirable embodiment, the compounds inhibit production of amyloidprecursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in a cell or a mammal by atleast 30, 50, 60, 70, 80, 90, 95, or 100% compared to a buffer control,as measured using standard assays such as those described herein. Inanother desirable embodiment, the compound inhibits production ofamyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in a cell or a mammalby at least 2, 5, 10, 20, or 50-fold compared to a buffer control, asmeasured using standard assays such as those described herein.

As used herein, “contacting” means exposure of at least one cell to acompound of the present invention. The cell of this invention can be,but is not limited to, a neural cell or supporting cell (e.g., glial orastrocyte). The term “neural cell” is defined as any cell that can belocated in the central or peripheral nervous system or is a precursor orderivative thereof, including, for example, but not limited to, neuronalcells, glial cells, neural stem cells, neuronal stem cells andneuroblasts. The cell can be contacted in vitro with the compound, forexample, by adding the compound to the culture medium (by continuousinfusion, by bolus delivery, or by changing the medium to a medium thatcontains the compound), or the cell can be contacted with the compoundin vivo (e.g., by local delivery, systemic delivery, intravenousinjection, bolus delivery, or continuous infusion). In vitro contact maybe preferred, for example, for measuring the effect of the compound on apopulation of cells. In vivo contact would be employed for inhibitingproduction of amyloid precursor protein in a subject in need of suchinhibition, (e.g., a subject) with a neurodegenerative disease, forexample, Alzheimer's Disease.

The subject of this invention can be any mammal that produces amyloidprecursor protein, such as a primate and more desirably, a human. Thesubject of this invention can also be domesticated animals, such ascats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats,etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig,etc.).

The duration of contact with a cell or population of cells is determinedby the time the compound is present at physiologically effective levelsor at presumed physiologically effective levels in the medium orextracellular fluid bathing the cell or cells. Desirably, the durationof contact is 1-48 hours and, more desirably, for 24 hours, but suchtime would vary based on the half-life of the compound and could beoptimized by one skilled in the art using routine experimentation.

Examples of compounds used in the methods of this invention forinhibiting amyloid protein precursor include, but are not limited to,(+)-phenserine, (+)-cymserine, (+)-N¹-phenethylnorcymserine, or(+)—N¹,N⁸-bisnorcymserine. In a desired embodiment, the compound is(+)-phenserine, compound V, wherein X and Y are NCH₃, R₃ and R₈ aremethyl, and the compound is the substantially pure (+)-enantiomer, orcompound VII, wherein X is NR₅, where R₅ is benzyl, R₆ is (CH₂)₂N(CH₃)₂,and R₈ is methyl.

In another embodiment, the present invention also provides a method ofinhibiting production of amyloid precursor protein in a subject,comprising administering to the subject an effective amount of acompound having the structure I-XVI and any combination thereof in apharmaceutically acceptable carrier, whereby the compound inhibitsproduction of amyloid precursor protein in the subject.

In a desirable embodiment, the compounds of the invention inhibitproduction of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in thesubject or in a sample from the subject by at least 30, 50, 60, 70, 80,90, 95, or 100% compared to a buffer control, as measured using standardassays such as those described herein. In another desirable embodiment,the compound inhibits production of amyloid precursor protein, Aβ₁₋₄₀,and/or Aβ₁₋₄₂ in the subject or in a sample from the subject by at least2, 5, 10, 20, or 50-fold compared to a buffer control, as measured usingstandard assays such as those described herein.

The compounds of the present invention can be administered in vivo to asubject in need thereof by commonly employed methods for administeringcompounds in such a way to bring the compound in contact with cells. Thecompounds of the present invention can be administered orally,parenterally, transdermally, extracorporeally, topically or the like,although oral or parenteral administration is typically desired.Parenteral administration of the compounds of the present invention, ifused, is generally characterized by injection. Injectables can beprepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. As used herein, “parenteraladministration” includes intradermal, subcutaneous, intramuscular,intraperitoneal, intravenous, intra-articular and intratracheal routes.Additionally, the compound can be administered via a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference hereinin its entirety. The compounds can also be administered using polymerbased delivery systems, including, for example, microencapsulation,which techniques are well known in the art.

The dosage of the compound varies depending on the weight, age, sex andcondition of the subject as well as the method and route ofadministration. As an example, the dosage of the compound is from about0.1 mg/kg to about 100 mg/kg of body weight. The lower limit for thedosage can be about 0.1, 0.5, 1, 2, 5, 10, 15, 20, 25, 30, or 40 mg/kgand the upper limit can be about 10, 15, 20, 25, 30, 40, 50, 60, 70, 80,90, or 100 mg/kg. Any lower limit can be used with any upper limit. Moredesirably, the compound is administered in vivo in an amount of about 1to about 20 mg/kg. Thus, an administration regimen could includelong-term, daily treatment. By “long-term” is meant at least two weeksand, desirably, several weeks, months, or years of duration. Necessarymodifications in this dosage range may be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed.,latest edition), Mack Publishing Co., Easton, Pa. The dosage can also beadjusted by the individual physician in the event of any complication.

The compounds can be administered conventionally as compositionscontaining the active compound as a predetermined quantity of activematerial calculated to produce the desired therapeutic effect inassociation with the required diluent (i.e., carrier or vehicle).Depending on the intended mode of administration, the compound can be inpharmaceutical compositions in the form of solid, semi-solid or liquiddosage forms, such as, for example, tablets, suppositories, pills,capsules, powders, liquids, suspensions, lotions, creams, gels, or thelike, desirably in unit dosage form suitable for single administrationof a precise dosage. The compositions will include, as noted above, aneffective amount of the selected compound in combination with apharmaceutically acceptable carrier and, in addition, may include othermedicinal compounds, pharmaceutical compounds, carriers, adjuvants,diluents, etc. By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected compound withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc. an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying compounds, pH bufferingcompounds and the like, for example, sodium acetate, sorbitanmonolaurate, triethanolamine sodium acetate, triethanolamine oleate,etc. Thus, the compositions are administered in a manner compatible withthe dosage formulation and in a therapeutically effective amount. Asdiscussed above, precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner and are peculiarto each individual.

For oral administration, fine powders or granules may contain diluting,dispersing, and/or surface active compounds, and may be presented inwater or in a syrup, in capsules or sachets in the dry state, or in anonaqueous solution or suspension wherein suspending compounds may beincluded, in tablets wherein binders and lubricants may be included, orin a suspension in water or a syrup. Where desirable or necessary,flavoring, preserving, suspending, thickening, or emulsifying compoundsmay be included. Tablets and granules are desired oral administrationforms, and these may be coated.

In another embodiment, the present invention provides a method oftreating a disorder associated with abnormal production of amyloidprecursor protein, such as, for example, dementia in a subject,comprising administering to the subject an effective amount of thecompound having the formula I-XVI and any combination thereof in apharmaceutically acceptable carrier, whereby the compound treats thedisorder in the subject. As used herein, the term “dementia” describes aneurodegenerative disorder that results from an organic brain disease inwhich a subject experiences usually irreversible deterioration ofintellectual faculties with accompanying emotional disturbances. Anexample of dementia includes, but is not limited to, Alzheimer'sdisease. An example of another disorder that can be treated by themethods of this invention includes, but is not limited to, cerebralamyloidosis. In a desirable embodiment, a compound used for thetreatment of dementia improves a symptom associated with dementia orAlzheimer's, stabilizes a symptom, or delays the worsening of a symptom.In other desirable embodiments, the compound increases the life-span ofa subject compared to the average life-span of corresponding subjectsnot administered the compound. In yet other desirable embodiments, thecompound is used to prevent or delay the onset of dementia orAlzheimer's.

In general, an “effective amount” of a compound is that amount needed toachieve the desired result or results. Thus, for example, administeringto a subject (e.g., a human) with Alzheimer's disease an effectiveamount of a compound of the present invention can result in slowing,stopping, or even possibly reversing the deterioration of the subject'sintellectual faculties and other accompanying neurological signs andsymptoms. Therefore, the inhibition of the production of amyloidprecursor protein, by the methods of the present invention, treats thesubject with Alzheimer's disease. The effective amount of the compoundneeded to treat dementia is from about 0.5 mg to about 200 mg. The lowerlimit for the effective amount of the compound can be about 0.5, 1, 2,5, 10, 20, 30, 40, 50, 100, or 150 mg, and the upper limit can be about50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200 mg. Any lower limit can be used with any upper limit. In oneembodiment, when the subject is a human, the effective amount ofcompound to treat dementia is from about 0.5 to about 100 mg. In anotherembodiment, (+)-phenserine, (+)-cymserine, (+)—N¹-phenethylnorcymserine,(+)—N¹,N⁸-bisnorcymserine, compound V, wherein X and Y are NCH₃, R₃ andR₈ are methyl, and the compound is the substantially pure(+)-enantiomer, or compound VII, wherein X is NR₅, where R₅ is benzyl,R₆ is (CH₂)₂N(CH₃)₂, and R₈ is methyl can be used in these amounts totreat dementia. In a desired embodiment, (+)-phenserine can be used inthese amounts to treat dementia in a subject.

In a further embodiment, the present invention relates to a method ofbinding an amyloid precursor protein messenger RNA 5′ untranslatedregion (5′UTR) in a cell, comprising contacting the cell with a compoundhaving the formula I-XVI and any combination thereof, whereby thecompound binds the amyloid precursor protein messenger RNA 5′untranslated region in the cell, thereby inhibiting amyloid proteinproduction. The amyloid precursor protein messenger RNA 5′UTR conferstranslational control of βAPP protein synthesis. In one embodiment,(+)-phenserine, (+)-cymserine, (+)-N¹-phenethylnorcymserine,(+)—N¹,N⁸-bisnorcymserine, compound V, wherein X and Y are NCH₃, R₃ andR₈ are methyl, and the compound is the substantially pure(+)-enantiomer, or compound VII, wherein X is NR₅, where R₅ is benzyl,R₆ is (CH₂)_(n)N(CH₃)₂, and R₈ is methyl can be used to bind the amyloidprecursor protein with messenger RNA 5′ UTR. In a desired embodiment,(+)-phenserine can be used to bind to an amyloid precursor proteinmessenger RNA 5′ untranslated region in a cell. In a desirableembodiment, at least 30, 50, 60, 70, 80, 90, 95, or 100% of the amyloidprecursor protein mRNA in a cell is bound by the compound.

In a further embodiment, the present invention relates to a method ofinhibiting translation of an amyloid precursor protein messenger RNA ina cell, comprising contacting the cell with a compound having theformula I-XVI and any combination thereof, whereby the compound bindsthe amyloid precursor protein messenger RNA 5′ and/or 3′ untranslatedregion in the cell, or binds a protein that interacts with the amyloidprecursor protein messenger RNA 5′ and/or 3′ untranslated region in thecell, or alters a process, either indirectly or directly, such asglycosylation or phosphorylation that then changes the binding of aspecific regulatory protein to amyloid precursor protein messenger RNA5′ and/or 3′ untranslated region in the cell, thereby inhibiting amyloidprotein production. In one embodiment, (+)-phenserine, (+)-cymserine,(+)-N¹-phenethylnorcymserine, (+)—N¹,N⁸-bisnorcymserine, compound V,wherein X and Y are NCH₃, R₃ and R₈ are methyl, and the compound is thesubstantially pure (+)-enantiomer, or compound VII, wherein X is NR₅,where R₅ is benzyl, R₆ is (CH₂)₂N(CH₃)₂, and R₈ is methyl can be used toinhibit the translation of the amyloid precursor protein with messengerRNA. In a desired embodiment, (+)-phenserine can be used to inhibit thetranslation of the amyloid precursor protein messenger RNA byinterfering with the post-transcriptional regulation of the amyloidprecursor protein RNA in a cell. In a desirable embodiment, the compoundinhibits production of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂by at least 30, 50, 60, 70, 80, 90, 95, or 100% compared to a buffercontrol, as measured using standard assays such as those describedherein. In another desirable embodiment, the compound inhibitsproduction of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ by atleast 2, 5, 10, 20, or 50-fold compared to a buffer control, as measuredusing standard assays such as those described herein.

The present invention also provides a method of screening a compound forthe ability to inhibit production of amyloid precursor protein, Aβ₁₋₄₀,and/or Aβ₁₋₄₂, comprising (a) contacting the cell with the compoundhaving the formula I-XVI and any combination thereof, and (b) detectinga decrease in amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂production in a cell contacted with the compound as compared to theamount of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ production ina control cell not contacted with the compound, whereby decreasedproduction of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in thecell identifies the compound as having the ability to inhibit theproduction of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in acell. As shown in the Examples section below, a person of skill in theart can measure the amount of βAPP, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ production ina control population of cells and compare the production of βAPP,Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in a population of cells contacted with a compoundto be screened by the methods of the present invention. A decrease inthe production of βAPP, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ in a population of cellscontacted with a compound as compared to the production of βAPP, Aβ₁₋₄₀,and/or Aβ₁₋₄₂ in a population of control cells identifies the compoundas having the ability to inhibit production of amyloid precursorprotein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂. In a desirable embodiment, the compoundinhibits production of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂by at least 30, 50, 60, 70, 80, 90, 95, or 100% compared to a buffercontrol, as measured using standard assays such as those describedherein. In another desirable embodiment, the compound inhibitsproduction of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ by atleast 2, 5, 10, 20, or 50-fold compared to a buffer control, as measuredusing standard assays such as those described herein.

The present invention further provides a method of screening a compoundfor the ability to inhibit amyloid precursor protein production bybinding an amyloid precursor protein messenger RNA 5′ untranslatedregion, comprising (a) contacting the messenger RNA with the compound;(b) detecting the binding of the compound to the amyloid precursorprotein-messenger RNA 5′ untranslated region; and (c) detecting theinhibition of amyloid precursor protein production from an amyloidprecursor protein-messenger RNA 5′ untranslated region, therebyidentifying a compound having the ability to inhibit amyloid precursorprotein messenger RNA 5′ untranslated region. The binding of thecompound to the amyloid precursor protein messenger RNA 5′ untranslatedregion inhibits β amyloid precursor protein (βAPP) from the messengerRNA by directly preventing the binding of the ribosomal translationalsubunit with the mRNA through steric hindrance. The detection of bindingof a compound to the 5′ UTR of the amyloid precursor protein messengerRNA can be carried out by methods standard in the art for detecting thebinding of substances to nucleic acids such as RNA. The detection ofinhibition of amyloid precursor protein production upon contact with thecompound can be carried out by the methods provided in the Examplesherein, as well as protocols well known in the art. The messenger RNAcan be in a cell or in a cell-free environment (e.g., a cell-freetranslation system). In a desirable embodiment, at least 30, 50, 60, 70,80, 90, 95, or 100% of the amyloid precursor protein mRNA in a cell isbound by the compound.

The present invention further provides a method of screening a compoundfor the ability to inhibit amyloid precursor protein, Aβ₁₋₄₀, and/orAβ₁₋₄₂ production by inhibiting translation of the amyloid precursorprotein messenger RNA, comprising (a) contacting the cell with thecompound; and (b) detecting the inhibition of amyloid precursor protein,Aβ₁₋₄₀, and/or Aβ₁₋₄₂ production from an amyloid precursor protein RNA,thereby identifying a compound having the ability to inhibit amyloidprecursor protein messenger RNA translation. In another embodiment, thescreening method further comprises after step (a) detecting the amountof the amyloid precursor protein-messenger RNA. In one embodiment, theamount of amyloid precursor protein mRNA is not inhibited by thecompound or is inhibited by less than 80, 80, 50, 40, 30, 20, or 10%. Inthis case, the compound primarily or only inhibits the translation ofamyloid precursor protein. It is also contemplated that in otherembodiments a compound may inhibit both transcription and translation ofamyloid precursor protein or inhibit only transcription of amyloidprecursor protein. While not meant to limit the invention to anyparticular mechanism of action, the inhibition of translation by thecompound can result from the binding of the compound to the amyloidprecursor protein messenger RNA 5′ and/or 3′untranslated region(s)inhibiting β amyloid precursor protein (βAPP) from the messenger RNA bydirectly preventing the binding of the ribosomal translational subunitwith the mRNA through steric hindrance or by inhibiting the binding ofan important regulatory protein, or the binding of the compound to theregulatory protein or by the compound modifying an important regulatoryprotein such that it no longer can interact with the amyloid precursorprotein RNA. The direct detection of binding of a compound to the 5′and/or 3′ UTR(s) of the amyloid precursor protein messenger RNA can becarried out by methods standard in the art for detecting the binding ofsubstances to nucleic acids such as RNA, such as NMR or massspectroscopy. The detection of inhibition of amyloid precursor proteinproduction upon contact with the compound can be carried out by themethods provided in the Examples herein, as well as protocols well knownin the art. The messenger RNA can be in a cell or in a cell-freeenvironment (e.g., a cell-free translation system). In a desirableembodiment, the compound inhibits production of amyloid precursorprotein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ by at least 30, 50, 60, 70, 80, 90, 95,or 100% compared to a buffer control, as measured using standard assayssuch as those described herein. In another desirable embodiments thecompound inhibits production of amyloid precursor protein, Aβ₁₋₄₀,and/or Aβ₁₋₄₂ by at least 2, 5, 10, 20, or 50-fold compared to a buffercontrol, as measured using standard assays such as those describedherein. In yet another desirable embodiment, at least 30, 50, 60, 70,80, 90, 95, or 100% of the amyloid precursor protein mRNA in a cell isbound by the compound. In still another embodiment, at least 30, 50, 60,70, 80, 90, 95, or 100% of the compound is directly or indirectly boundto an amyloid precursor protein messenger RNA 5′ or 3′ untranslatedregion or to an RNA binding protein that interacts with the amyloidprecursor protein messenger RNA 5′ or 3′ untranslated region.

In another embodiment, the invention relates to a method of screening acompound for the ability to inhibit amyloid protein production byeliciting a change in reporter gene expression, comprising:

-   (a) contacting a cell transfected with a reporter gene containing    the 5′ and/or 3′ UTR(s) of the amyloid precursor protein messenger    RNA with the compound, and-   (b) detecting a decrease in reporter gene expression or activity;    thereby identifying a compound having the ability to inhibit amyloid    precursor protein messenger RNA translation.

In a desirable embodiment, the compound inhibits reporter geneexpression or activity by at least 30, 50, 60, 70, 80, 90, 95, or 100%compared to a buffer control, as measured using standard assays such asthose described herein. In another desirable embodiment, the compoundinhibits reporter gene expression or activity by at least 2, 5, 10, 20,or 50-fold compared to a buffer control, as measured using standardassays such as those described herein.

The compounds used in the screening methods of this invention can be,but is not limited to, a compound having the structure I-XVI and anycombination thereof. In various embodiments, the compound is a(+)-isomer, (−)-isomer, or a racemic mixture. In various embodiments ofany of the methods of the invention, the compound is a (+)-isomer,(−)-isomer, or a racemic mixture of MES 9295 (FIG. 13E). In otherembodiments of any of the methods of the invention, the compound is nota (+)-isomer, (−)-isomer, or a racemic mixture of MES 9295.

In desirable embodiment of any of the aspects of the invention, thecompound inhibits cholinesterase activity, such as acetylcholinesteraseor butyrylcholinesterase activity, by less than 80, 70, 60, 50, 40, 30,20, 10, or 5% (in order of increasing preference) relative to a bufferonly control. In other desirable embodiments, inhibition ofcholinesterase activity, such as acetylcholinesterase orbutyrylcholinesterase activity, by the compound is at least 2, 5, 10,20, 50, or 100-fold less than the inhibition of cholinesterase activityby the corresponding amount of (−)-phenserine. In other desirableembodiments, inhibition of cholinesterase activity, such asacetylcholinesterase or butyrylcholinesterase activity, by a (+) isomeror a racemic mixture is at least 2, 5, 10, 20, 50, or 100-fold less thanthe inhibition of cholinesterase activity by the corresponding amount of(−)-isomer. In yet other desirable embodiments, the compound issubstantially free of cholinesterase inhibitory activity. Inhibition ofcholinesterase activity can be measured using any standard assay. Forexample, the assay and the in vivo mouse model described in U.S. Pat.No. 4,791,107, which is incorporated by reference in its entirety, orthe in vivo mouse model described herein can be used.

In other desirable embodiments of any of the aspects of the invention,the compound results in a less than 20, 10, 5, or 2-fold increase in theamount of released lactate dehydrogenase (a marker of cell viability andintegrity) relative to the amount of released lactate dehydrogenase inthe absence of the compound or in the presence of a buffer control. Instill other desirable embodiments, the amount of compound that isadministered to a subject per kg body weight of the subject does notcause tremors or death when administered in the in vivo mouse modeldescribed herein. In yet other desirable embodiments, less than 80, 70,60, 50, 40, 30, 20, 10, or 5% of the neuronal cells contacted with thecompound are killed by the compound. In another desirable embodiment,the does of the compound is equal to or greater than 1 mg/kg of bodyweight, 5 mg/kg, or 10 mg/kg.

In other desirable embodiments of any of the aspects of the invention,the compound inhibits production of amyloid precursor protein, Aβ₁₋₄₀,and/or Aβ₁₋₄₂ by at least 30, 50, 60, 70, 80, 90, 95, or 100% comparedto a buffer control. In another embodiment, the compound inhibitsproduction of amyloid precursor protein, Aβ₁₋₄₀, and/or Aβ₁₋₄₂ by atleast 30, 50, 60, 70, 80, 90, 95, or 100% compared to a buffer control,and inhibits cholinesterase activity by less than 80, 70, 60, 50, 40,30, 20, 10, or 5% relative to a buffer only control. In other desirableembodiments, the compound inhibits intracellular and/or extracellularAPP or Aβ production. In yet other desirable embodiments, the compoundinhibits production of amyloid precursor protein in a cell or mammal byat least 2, 5, 10, 20, or 50-fold more than it inhibits cholinesteraseactivity in the cell or mammal. In still other desirable embodiments,the amount of compound required to inhibit production of amyloidprecursor protein in a cell or mammal by 50% (IC₅₀ value) is at least 2,5, 10, 20, 50, or 100-fold less than the amount of compound required toinhibit cholinesterase activity by 50% (IC₅₀ value) in the cell ormammal, as measured using standard assays.

In other desirable embodiments of any of the methods of the invention,the compound is(+)-3,3a,8,8a-Tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-olmethyl ether;(+)-3,3a,8,8a-Tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol;(+)-3,3a,8,8a-tetrahydro-3a, 8-dimethyl-2H-thieno-[2,3-b]indole-5-olbutyl carbamate; (+), -3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol heptylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a, 8-dimethyl-2H-thieno[2,3-b]indole-5-olphenylcarbamate; (+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl;-2H-thieno[2,3-b]indole-5-ol 2′-methylphenylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol2′ethylphenylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol2′-isopropylphenylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol4′-isopropylphenylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol2′,4′-dimethylphenylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-olN,N-dimethylcarbamate; (+)-O-methyl-N(1)-noreseroline;(+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-olmethyl ether; (+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5 ol ((−)-thiaphysovenol);(+)-3,3a,8,8a-Tetrahydro-3a, 8-dimethyl-2H-thieno[2,3-b]indole-5-ol2′,4′-dimethylphenylcarbamate; (+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol 2′-methylphenylcarbamate;(+)-3,3a,8,8a-tetrahydro-3a, 8-dimethyl-2H-thieno[2,3-b]indole-5-ol4′-isopropylphenylcarbamate; (+)-3,3a,8,8a-tetrahydro-3a,8-dimethyl-2H-thieno[2,3-b]indole-5-ol 4′-isopropylphenylcarbamate, or amixture of the (+)- and (−)-enantiomers thereof (e.g., a racemicmixture). In other embodiments, the compound is the (+)-enantiomer or amixture of the (+)- and (−)-enantiomers thereof (e.g., a racemicmixture) of a compound disclosed in column 2, line 16 through column 3,line 40 of U.S. Pat. No. 5,378,723 (Brossi et al., issued Jan. 3, 1995).

In other desirable embodiments of any of the methods of the invention,the compound is a compound that falls with the general formula (e.g.,column 1, lines 11-52) and or a specifically disclosed compound (e.g., acompound listed in column 21, line 59 through column 38, line 42) inU.S. Pat. No. 4,791,107 (Hamer et al., issued Dec. 13, 1988), and thecompound has negligible cholinesterase inhibitory activity (e.g., causesno detectable cholinesterase inhibition or causes less than 50, 40, 30,20, 10, or 5% inhibition). In other desirable embodiments,administration of the compound produces no adverse side-effects due toinhibition of cholinesterase activity. These compounds can be the (+)-or (−)-isomers or a mixture thereof (e.g., a racemic mixture). In otherembodiments, the compound is a compound that falls with the generalformula and or a specifically disclosed compound in U.S. Pat. No.4,791,107, and the compound is administered in a dose of at least 1mg/kg, 5 mg/kg, or 10 mg/kg.

In other desirable embodiments of any of the methods of the invention,the compound is a compound that falls with a general formula disclosedin U.S. Pat. No. 5,378,723 (Brossi et al., issued Jan. 3, 1995), U.S.Pat. No. 5,171,750 (Brossi et al., issued Dec. 15, 1992), or U.S. Pat.No. 5,998,460 (Brossi et al., issued Dec. 7, 1999), and the compound hasnegligible cholinesterase inhibitory activity (e.g., causes nodetectable cholinesterase inhibition or causes less than 50, 40, 30, 20,10, or 5% inhibition) or is administered in a dose of at least 1 mg/kg,5 mg/kg, or 10 mg/kg. [As an alternative, these structures could be cutand pasted into the application.] In other desirable embodiments of anyof the methods of the invention, the compound is the (+)-isomer or aracemic mixture of a compound that falls with a general formula or isspecifically disclosed in U.S. Pat. No. 5,378,723, U.S. Pat. No.5,171,750, or U.S. Pat. No. 5,998,460.

In other embodiments of any of the methods of the invention, thecompound is not (−)-phenserine, (−)-physostigmine,(−)-heptyl-physostigmine, (−)-physovenine, (−)—N(1)-norphysostigmine,MES9217 (FIG. 13H), MES9299 (FIG. 13L), or MES9329 (FIG. 13M). In otherembodiments of any of the methods of the invention, the compound is nota compound disclosed in U.S. Pat. No. 5,378,723, U.S. Pat. No.5,171,750, U.S. Pat. No. 5,998,460, or U.S. Pat. No. 4,791,107.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds claimed herein are made and evaluated, and are intended to bepurely exemplary of the invention and are not intended to limit thescope of what the inventors regard as their invention. Efforts have beenmade to ensure accuracy with respect to numbers (e.g., amounts,temperature, etc.) but some errors and deviations should be accountedfor. Unless indicated otherwise, parts are parts by weight, temperatureis in ° C. and is at room temperature, and pressure is at or nearatmospheric.

General Considerations

Phenserine: Phenserine is a member of a family of compounds that arephenylcarbamates of hexahydropyrrol indoles with specific side groupsthat provide it selectivity against either acetyl- orbutyryl-cholinesterase, a high brain uptake and a long duration ofpharmacological action (Greig et al., 1995; Brossi et al., 1996). Thecompound was synthesized in its optically (>99.9%) and chemically(>99.9%) pure (−)- and (+)-enantiomeric forms as a tartrate salt, asdescribed (Yu and Brossi, 1988; Greig et al., 1995). The concentrationof compound required to inhibit 50% AChE activity was 22 nM for(−)-phenserine, whereas >25,000 nM was inactive for optically pure(+)-phenserine.Drug treatment: SK-N-SH neuroblastoma cells were cultured on 60 nmdishes at a concentration of 3×10⁶ cells, and SH-SY-5Y neuroblastoma andU373 astrocytoma cell lines were plated in 100 mm dishes at aconcentration of 3×10⁵ cells. The cells were allowed to grow in completemedium (10% FBS, 2 mM glutamine in DMEM) for 3 to 4 days until theyreached 70% confluence. To start the experiment, spent medium wasremoved and replaced with fresh medium (SKNSH—4 ml of DMEM+0.5% FBS;U373—5 ml of DMEM+2.5% FBS) containing 0, 5, or 50 μM phenserine. Thecells were incubated at 37° C., 5% CO₂ for the specific times indicated.Different media and sera were purchased from Life Technologies(Gaithersberg, Md.).Inhibitor treatment: One day prior to drug treatment, confluent culturesof U373 cells were pretreated with 25 nM of ERK specific inhibitor,PD98059 (Calbiochem-Novabiochem, La Jolla, Calif.), in 4.5 ml of 2.5%FBS, 2 mM glutamine and DMEM for 16 hours. Phenserine was added to eachassay plate and a final volume of 5 min was reached. To examine for PI 3kinase involvement, an active 2 μM concentration of the PI 3 kinaseinhibitor, LY294002 (Calbiochem-Novabiochem, La Jolla, Calif.), in 4.5ml of 2.5% FBS, 2 mM glutamine and DMEM was added to each assay plateand incubated for 30 minutes prior to the addition of phenserine.Appropriate vehicle controls were run alongside treated samples.Lysate Preparation: At each time point, the spent medium was collectedand stored at −70° C. for later analysis of secretory βAPP levels. Thecells were washed twice with PBS, pH 7.4 and incubated on ice for 15 minfor lysis with 100 μL of lysis buffer (20 mM HEPES, 2 mM EGTA, 50 mMβ-glycophosphate, 1 mM sodium orthovanadate, 1% Triton X-100, 10%glycerol) containing appropriate protease inhibitors (2 mM PMSF, 100μg/mL aprotinin, 25 μM leupeptin and 20 μg/nL soybean trypsininhibitor). Each lysate was microcentrifuged for 15 min at 14 000 rpm.Protein levels of the supernatant were analyzed by the Bradford proteinassay (BioRad, Melville, N.Y.).Western Blot: Fifteen μg of protein from each sample was mixed with theappropriate volume of 5× Laemmli buffer and boiled for 5 min at 100° C.The samples were loaded onto a 10% NuPAGE Bis-Tris gel in 1× NuPAGE MOPSSDS running buffer (NOVEX, San Diego, Calif.) and the proteins separatedat 200 V for 45 min. The gels then were transferred onto nitrocelluloseat 25 V for 1.5 h. The blots were blocked with 5% non-fat dry milk in 10mM Tris, pH 8.0 containing 150 mM NaCl for 1 h and washed twice for 15min in large volumes of TBST (10 mM Tris, pH 8.0, 150 mM NaCl and 0.05%Tween 20). Each blot was probed for 2 h with either 22C11anti-βAPP-terminal antibody (Boehringer Mannheim, Indianapolis, Ind.),diluted to a concentration of 2.5 μg/mL or anti-activated ERK antibody(Promega, Madison, Wis.), diluted to a concentration of 25 ng/mL. Theblots were washed twice for 15 min in TBST and placed in secondaryantibody, anti-mouse IgG- or anti-rabbit IgG conjugated to horse radishperoxidase (Sigma, St. Louis, Mo.), for 30 min. Three final TBST washesof 20 min duration each were performed before the samples were detectedby chemiluminesence and exposed to film, as per the manufacturer'sinstructions Amersham Life Science Inc., Arlington Heights, Ill.).Additionally, all blots also were stained with Ponceau S (Sigma, St.Louis, Mo.) to determine equivalent loading of samples. Densitometricquantification of blots was undertaken by using a CD camera andNIH-IMAGE (version 4.1).Lactate Dehydrogenase Assay: Measurement of released lactatedehydrogenase (LDH) in the conditioned medium was undertaken as a markerof cell viability and integrity, as described previously (Lahiri et al.,1997 and 1998).Total Aβ Assay: Total Aβ peptide levels in SH-SY-5Y and SK-N-SH culturedsamples were assayed by a sensitive ELISA (Suzuki et al., 1994). Fortotal Aβ measurements in conditioned medium, the rabbit polyclonalantibody #3160 (1-40 residues of Aβ) was used as a capture antibody forall species of Aβ peptide (Aβ1-40 and Aβ1-42) while monoclonal antibody4G8 (17-25 residues of Aβ) was used to detect Aβ peptide levels, and thevalues were expressed as the mean of six independent assays.Transfection: One day prior to transfection, U373 cells were plated onto100 mm dishes at a density of 3×10⁵ cells. On the day of transfection,the cells were given 5 ml of fresh media containing 10% FBS, 2 mMglutamine in DMEM. The cells were transfected using a calcium phosphateprecipitation method, as per the manufacturer's protocol and describedpreviously (Rogers et al., 1999). Briefly, for each plate, three μg ofDNA (5′UTR APP-PSV2-CAT or PSV2-CAT vector) were placed in a finalvolume of 500 μL of 250 mM CaCl₂. The chloramphenicol acetyl transferase(CAT) gene was used as a reporter gene. The DNA solution was slowlypipetted into an aerated, equivalent volume of 2×HeBS, pH 7.05. Theresulting precipitate was allowed to stand 10-20 min at RT before itsaddition to the cells. After 18 h, the medium was changed and thetransfected cells were left for two days before drug treatment.CAT Assay: The cell lysates from transfected U373 cells treated withphenserine were analyzed for their CAT activity using a calorimetricenzyme immunoassay (Boehringer Mannheim, Indianapolis, Ind.). Briefly,50 μg of protein (an amount previously found to lie within the linearrange of the assay) were placed onto anti-CAT coated microtiter platemodules and allowed to bind for 1 h at 37° C. The plates were washedthoroughly after each step. Next, a digoxigenin-labeled anti-CATantibody was added to the samples and incubated for 1 h at 37° C. Asubsequent antibody, anti-digoxigenin conjugated to peroxidase, wasplaced in the wells for another hour under similar conditions. Finallyperoxidase substrate, ABTS, was added to each well and the absorbance ofeach sample was measured at a wavelength of 405 nm.Northern Blotting: Total RNA (10 μg) was extracted and prepared fromtreated astrocytoma cells using an RNA-STAT kit (Tel-test, Friendswood,Tex.). The samples were denatured in form amide, MOPS buffer,formaldehyde, dye mix and ethidium bromide at 65° C. for 10 min, placedon ice for 5 min and electrophoresed on a 1.0% agarose-formaldehyde gel.The gel was blotted onto Hybond Nitrocellulose filters and immobilizedby UV crosslinking and heating filters for 2 hours. Each filter wasprehybridized in hybridization buffer (1% BSA, 7% SDS, 0.5 M phosphatebuffer, pH 7, 1 mM EDTA) for at least 2 hours. The filter was hybridizedovernight with probe. Following hybridization, the filters were washedtwice with wash solution containing 0.5% BSA, 5% SDS, 40 mM phosphatebuffer, pH 7, 1 mM EDTA for 30 min at 65° C. The βAPP cDNA probecorresponded to a unique internal BglII/SpeI fragment generated fromhuman KNAPP cDNA (provided by John Kusiak, Gerontology Research Center,IRP, NIA, NIH). Equal loading of samples was verified by rehybridizingthe filter with a human actin gene using an actin β-cDNA probe (ClontechLaboratories, Palo Alto, Calif.).Plasmid Constructs: The plasmid PSV2-APP-CatD was provided by Dr. Rogers(1999). Briefly, the pSV₂(APP)CAT construct was generated by inserting a90 bp fragment of the βAPP gene 5′-UTR immediately upstream of the CATgene into the pSV₂ vector.Statistics: A two-tailed Student's t-test was carried out to compare twomeans. When more than two means were compared, one-way analysis ofvariance, together with a Bartlett's test for homogeneity of variancesand a Dunnett's multiple comparison test were used. The level ofsignificance was defined as P<0.05.Phenserine Decreases βAPP and AD Levels in Neuroblastoma Cells

βAPP protein levels were measured after treatment of the SH-SY-5Y cellswith 5 μM (+)-phenserine and (−)-phenserine for 0.5, 1, 2, and 4 hours(FIG. 3). SH-SY5Y cells were incubated with 5 μM (+)-phenserine or (−)phenserine for 0, 0.5, 1, 2 and 4 hours to determine the effect of thedrug on βAPP protein levels. Western blots of cell lysates containing 15μg of total protein per lane were analyzed. The blot was sectioned intotwo halves and the top portion of the blot was probed with an N-terminaldirected anti-βAPP antibody whereas the remaining blot was probed withan antibody directed to phosphorylated ERK. In accord with previousreports (Lahiri et al., 1997, 1998; Waskiewics and Cooper, 1995), twohigh molecular weight bands corresponded to alternate forms of βAPP(100-125 kDa) and ERK ½ (42-46 kDa). The stereoisomeric forms of thedrugs have opposite affects on cholinesterase activity: (+)-phenserineexhibits no anti-cholinesterase activity whereas (−)-phenserine haspotent enzymatic activity (Greig et al., 1995; Brossi et al., 1996). Inboth experiments, the βAPP levels in the cell lysates slowly decreasedat each time point with the most dramatic decline observed after 4hours. During this period, the cells were also examined for theirability to induce signal transduction pathways. Mitogen stimulatedkinase, ERK1/2, was detected in the treated samples at all times andpeaked at the 30 minute to 1 hour period. Stress activated transcriptionkinases, p38 and JNK, were not detected in the samples. Furthermore,media samples were analyzed for levels of Aβ at 4 hours and,additionally, at 8 and 16 hours to assess whether or not decline in βAPPtranslated into a decline in total Aβ peptide levels. Levels of Aβ werebelow detectable levels in both control and (−)-phenserine treatedcells. As a consequence, studies were repeated with SK-N-SH cells with(−)-phenserine and (+)-phenserine, which was used in all subsequentstudies unless otherwise indicated.

SK-N-SH cells were incubated with either (−)-phenserine (FIG. 4) or with(+)-phenserine (FIG. 5) for up to 16 hours. FIG. 4 illustrates theeffect of (−)-phenserine on βAPP protein levels (FIGS. 4A and 4B), LDHlevels (FIG. 4C) and total Aβ levels (FIG. 4D). βAPP levels are shown asa percent of controls after pretreatment with 0.5, 5 and 50 μM(−)-phenserine for 4, 8 and 16 hours (*significantly different fromcontrol, p<0.05). Western blots of conditioned media (FIG. 4A) and celllysates (FIG. 4B) were probed with a N-terminal directed anti-βAPPantibody. Following phenserine treatment of SK-N-SH cells for 16 hours,βAPP levels were reduced in a time- and concentration-dependent mannerin both conditioned media (FIG. 4A) and cell lysates (FIG. 4B).

LDH levels were measured in media from cells treated with and without 50μM (−)-phenserine for up to 16 hours. There was no significantdifference between treated and untreated levels up to 16 hours (p>0.05).This was not associated with cellular dysfunction, as determined bymeasurement of LDH levels versus untreated controls (FIG. 4C).

Quantification of levels of total Aβ was undertaken at 8 and 16 hoursand results shown in FIG. 4D demonstrate a (−)-phenserine inducedreduction of 14% and 31% (p<0.002), respectively, versus untreatedcontrols. (+)-Phenserine possessed a similar concentration- andtime-dependent action on βAPP levels. The concentration of total Aβpeptide was measured in media from SK-N-SH cells that were incubatedwith 50 μM (−)-phenserine for up to 16 hours. Levels fell from 6.95 to5.95 μM (14%) at 8 hours, and from 11.75 to 8.1 μM (31%, p<0.02) at 16hours in control and (−)-phenserine treated cell, respectively (FIG.4D). Likewise, (+)-phenserine reduced βAPP protein levels and total Aβpeptide levels at 16 hours (FIG. 5). This was not associated withtoxicity, as assessed by measurement of cell number and viability (LDHlevels).

Phenserine Associated Decrease of βAPP Levels in Astrocytoma Cell LineU373 is Not Dependent on ERK Activation

Following an extended period of (−)-phenserine treatment, U373 cellsexhibited a similar pattern of decreased βAPP protein synthesis. FIG. 6is a representative of 4 experiments that showed that βAPP levelsgradually decreased between 1 and 8 hours of treatment. U373 MGastrocytoma cells were treated with (−)-phenserine to determine itseffect on βAPP protein levels. The addition of ERK inhibitor, PD98059,and PI 3 kinase inhibitor, LY294002, was carried out to ascertainwhether or not (−)-phenserine action on βAPP was directed through thesesignaling pathways. Western blots of lysates (15 μg per lane) of U373cells incubated with 50 μM of (−)-phenserine for 0, 0.5, 1, 4, 8, 24 and48 hours were analyzed. The blot was divided into two sections. On theleft panel, the blot was probed with anti-βAPP antibody and on the rightpanel, the blot was probed with anti-phosphorylated ERK antibody. After8 hours, a slow recovery of βAPP was detectable (FIG. 6A) but its levelwas still lower than in untreated cells. The activation of ERK1/2 peakedat the 30 minute time point and remained elevated at a low level for theremainder of the assay.

In order to determine whether or not ERK involvement was directlyrelated to phenserine treatment, the cells were pretreated with PD98059,a specific inhibitor of MAP kinase (FIG. 6B). U373 MG cells werepretreated with 25 nM PD98059 for 16 hours prior to (−)-phenserinetreatment. Lysates were analyzed by western blots as described above.Although ERK levels decreased significantly, the pattern of βAPP levelsinduced by phenserine remained largely similar to U373 cells treatedwith drug without PD98059. In all cases, βAPP levels were decreased byin excess of 25%, as determined by densitometric quantification.

Phenserine action on βAPP through ERK independent, phophoinositol 3kinase (PI 3 kinase) stimulation was also assessed. Treatment ofastrocytoma cells with phenserine and LY294002, a specific inhibitor ofPI 3 kinase, showed a similar pattern of βAPP levels when compared to(−)-phenserine alone treated cells (FIG. 6C). U373 MG cells werepretreated with 200 μM LY294002 for 1.5 hours prior to the addition of(−)-phenserine. The cell lysate of each sample was analyzed as describedabove. βAPP protein levels were reduced by in excess of 25% (p<0.05)with (−)-phenserine treatment in FIGS. 6A-C, as determined bydensitometric quantification.

In summary, these studies demonstrate that the action of (−)-phenserineto reduce βAPP protein and total Aβ peptide levels did not occur viaclassical cholinergic or neurotransmitter mediated mechanism, as hasbeen suggested by Buxbaum et al., (1990, 1992, 1994) and Nitsch et al.(1992, 1994). This was supported by two previously unreported lines ofevidence. First, studies with the (+)-enantiomer, (+)-phenserine, thatis devoid of anticholinesterase activity and hence cholinergic action,still reduced βAPP protein and total Aβ peptide levels (FIG. 5). Second,the actions of (−)-phenserine βAPP protein and total Aβ peptidepersisted after the classical pathways involved in cholinergicmodulation were blocked (FIG. 6). In addition, in separate studies, when50 uM concentrations of the classical anticholinesterase,(−)-physostigmine, were applied to SK-N-SH cells, no reduction or changein βAPP protein and total Aβ peptide levels was found.

To demonstrate that the actions on βAPP protein and Aβ peptide were notrestricted to enantiomers of phenserine, identical studies wereundertaken with both enantiomers of cymserine (compound 46 in Table 1for (+)-enantiomer) and with (−)—N¹,N⁸-bisnorcymserine and(−)-N¹-phenethylcymserine. Similar to (−)-phenserine, the (−)-enantiomerof cymserine possessed anticholinesterase action and the (+)-enantiomerwas devoid of it. As shown in FIGS. 7A and 7B, both enantiomers reduceβAPP protein levels. Similarly, N¹,N⁸-bisnorcymserine andN¹-phenethylcymserine reduced βAPP protein levels (FIGS. 7C and 7D). Ineach case, Aβ peptide levels were also reduced, and there was notoxicity (as assessed by cell number and viability, as assessed by LDHmeasurement). However, at higher concentrations of N¹,N⁸-bisnorcymserineand N¹-phenethylcymserine, the compounds are toxic.

Phenserine Decreases βAPP Protein Levels Through the Action of aTranslational Enhancer in the APP-mRNA 5′ Untranslated Region

A recent report identified a 90 nt element from the 146 nt 5′untranslated region (5′UTR) of the βAPP mRNA that is able to confer a 3fold IL-1 responsive gene expression to CAT reporter mRNAs inastrocytoma cells (Rogers et al., 1999). Interleukin-1 was able toinduce βAPP protein levels in the absence of increased βAPP mRNAsynthesis. Parallel experiments with (−)-phenserine were examined forits ability to regulate βAPP protein levels in an identical manner. U373MG astrocytoma cells were transfected with 3 μg of pSV₂ (APP) CATplasmid or the parental vector pSV₂ CAT. Each set of transfection plateswas left unstimulated or treated with 50 μM of phenserine for theexperimental times listed below.

FIG. 8A is a representative CAT assay that shows that (−)-phenserine isable to decrease the level of APP-mRNA 5′UTR enhancement to a CATreporter mRNA in pSV₂ (APP) CAT transfected astrocytoma cells. CATactivity was assessed from lysates of transfected cells treated with(−)-phenserine for 0, 0.25, 0.5 and 1 hour and 50 mg from each samplewas measured in duplicate for each assay. Quantitation of the foldstimulus of CAT gene activity conferred by the 5′ UTR βAPP-mRNA wasmeasured. ELISA readings of CAT expression were measured at 405 mm. A4-fold decrease after phenserine treatment was sustained after 1 hour.In control samples, pSV₂ CAT transfected cells exhibited no inhibitionby (−)-phenserine at all time points, indicating that the parentalvector was unresponsive to drug treatment. In other assays where thetime of treatment was extended to 48 hours, only a 2-fold decrease inCAT reporter mRNA was detected with phenserine treatment. However, evenin these extended assays, CAT activity in control cells remainedundisturbed, indicating that the drug's effects were specific for the 5′UTR of βAPP mRNA. The expression level of CAT in the control vector vs.the 5′UTR containing vector prior to (−)-phenserine treatment wassimilar.

(−)-Phenserine decreases the level of βAPP levels through the influenceof the βAPP-mRNA 5′UTR region. Western blot analysis of βAPP proteinlevels was performed on lysates of transfected cells treated with(−)-phenserine for 0, 0.25, 0.5 and 1 hour (FIG. 8B). The blot wasprobed with anti-βAPP antibody. βAPP protein levels in transfected U373astrocytoma cells treated with phenserine showed a similar pattern ofresults as with the CAT assay (FIG. 8A). The introduction of the CATreporter constructs increased the level of βAPP over the endogenousuntransfected cells, in accord with that reported by Rogers et al.(1999). However, after (−)-phenserine treatment, βAPP levels in pSV₂(APP) CAT transfected astrocytomas gradually approached levels seen inuntransfected and untreated cells. In contrast to these results,phenserine treatment of U373 cells did not affect βAPP mRNA levels.

FIG. 8C is a representative northern blot of U373 cells treated withphenserine for time points up to 48 hours. Phenserine does not affectthe steady state levels of βAPP-mRNA levels. Ten μg of RNA isolated fromuntransfected and transfected cells treated with (−)-phenserine for 0,1, 4, 24 and 48 hours were analyzed by northern blot. Phosphoimageranalysis revealed steady-state expression of βAPP-mRNA in each sample.The same filter was stripped and rehybridized with a labeled human actinprobe to standardize the loading differences in individual samples.Standardization of each sample to actin mRNA expression showedconsistent levels of βAPP mRNA without any major fluctuations indensitometry readings. Clearly, phenserine's action of βAPP protein isat the level of translation, as northern blot analysis of untransfectedand transfected cells show little differences in levels of mRNAtranscription.

In Vivo Studies-Toxicity of (−)-Phenserine vs. (+)-Phenserine

On administration of (−)-phenserine to rodents by the i.p. route (1ml/kg in 0.9% saline) a fine tremor is observed at a dose of 5 mg/kg.This persisted for an hour and is related to central cholinergicoverdrive. Tremor together with symptoms of peripheral cholinergicoverdrive (specifically, salivation and lacrimation) were seen at a doseof 7.5 mg/kg for some 3 hours. Animals were incapacitated at 20 mg/kg(N=3 per dose group), and 2 were killed when moribund. Animals (N=2)administered 20 mg/kg (+)-phenserine were without clinical symptoms andappeared normal.

Illustrated in FIG. 9 is the action of (−)-phenserine (2.5 mg/kg i.p.,once daily for 3 weeks) on brain cortex and CSF βAPP levels intransgenic mice (N=12) that significantly overexpress βAPP as aconsequence of the human Swedish βAPP mutation and mutant presenilin 1(Borchelt et al., 1997). (−)-Phenserine significantly reduced βAPPlevels by 55% in CSF and 10% in cerebral cortex. Greater reductionscould be achieved, but, as previously reported, doses of >5 mg/kgproduce cholinergic side effects for the (−)-enantiomer but not for the(+)-enantiomer.

Samples of cerebral cortex from the transgenic mice then were analyzedfor β-amyloid peptide (Aβ) levels. Specifically, Aβ₁₋₄₀ and Aβ₁₋₄₂levels, following formic acid extraction, were determined by ELISA assay(Suzuki et al., 1994). As shown in FIG. 10A, (−)-phenserine treatmentreduced Aβ₁₋₄₀ levels by 68% (p<0.05), whereas (−)-N¹-phenethylcymserinereduced levels by 54% (p<0.05). In contrast, (−)-phenserine reducedAβ₁₋₄₂ levels by 53% (p<0.05), compared to a 47% reduction (p<0.05)induced by (−)-N¹-phenethylcymserine, as shown in FIG. 10B.

Hence, even over as short a duration as three weeks in the life span oftransgenic mice (generally 18 to 24 months) that overexpress βAPP andoverproduce Aβ, daily phenserine and N¹-phenethylcymserineadministration, in well tolerated doses, reduce both βAPP and Aβ levels,thereby indicating that in vitro efficacy-translates to in vivoactivity.

In summary, the (+)-enantiomers of this invention are the focus of thepresent application. They are unnatural and totally synthetic compounds.The described studies demonstrated that both (+)- and (−)-enantiomerspossessed potent activity to reduce βAPP protein and total Aβ peptidelevels. However, the (+)-enantiomers are devoid of anticholinesteraseactivity, and hence lack cholinergic action. It is the cholinergicaction that is dose limiting with regard to the use of the(−)-enantiomers in vivo. The reductions in levels of βAPP proteindemonstrated in tissue culture studies occurred in two different typesof human neuroblastoma cell (SK-N-SH and SH-SY-5Y lines), as well as inastrocytoma cells (U 373 line). These in vitro effects translate into invivo activity as demonstrated herein.

Translational Effect of Phenserine on APP

1. Rate of APP Synthesis

8×10⁶ SHSY-5Y cells were plated on 100 mm dishes with DMEM containing10% FBS. After 36 hours, the culture medium was replaced with DMEMcontaining 0.5% FBS. The cells were incubated with low serum medium for1 hour. Thereafter, the medium was replaced with fresh low serum mediumwith and without 10 μM of phenserine for 16 hours.

After treatment (16 hrs) with and without (−)-phenserine (10 μM), thecells were incubated with methionine and cystine free DMEM containing 4mM of glutamine for 1 hour. After treatment with methionine and cystinefree medium, 2 ml of ³⁵S-labeled DMEM (100 μMCi/ml) with and withoutphenserine (10 μM) were added and incubated for 10 minutes. Thereafter,the labeled medium was carefully removed and the cells were suspended inlysis buffer containing with protease inhibitors and frozen at −80 C foruse in the immunoprecipitation assay.

APP protein was immunoprecipitated from 300 μg of total protein in eachsample using the polyclonal antibody O443, which recognizes a 20 aminoacid sequence in the carboxy terminal of APP, and protein A/G resinovernight at 4° C. Immunoprecipitated APP was eluted from the proteinA/G resin with 30 μl of elution buffer (10% beta-mercaptoethanol). Thesamples were loaded onto 10% trys-glycine gels, and the proteins wereseparated at 150 V for 90 min. The gels were fixed and dried at 80 C for60 min. The dried gels were exposed to Phosphor Screen (PACKARDInstrument Company, Inc., Meriden, Conn.) overnight and the βAPP signalswere quantitated on a phosphor imager. Phenserine significantlydecreased βAPP synthesis (52% reduction) without changing TCAprecipitable counts (FIG. 11A). This change in APP protein was notreflected in a change in mRNA levels as phenserine did not alter levelsof APP RNA (FIG. 11B).

2. Effect of Phenserine on Steady State APP Levels

3×10⁶ SK-N-SH cells were plated on 60 mm dishes with DMEM containing 10%FBS. After 36 hours, cells were incubated with low serum medium for 1hour. Thereafter, the medium was replaced with fresh low serum medium(0.5% FBS). To start the experiments, spent medium was removed andreplaced with fresh medium containing 0, 0.5, 5 or 50 μM of (+)- or(−)-phenserine. The cells were incubated with and without phenserine for16 hours. At the stated time (4, 8 and 16 hrs), 200 μl of medium wastransferred from each dish for assessment of extracellular APP levels.At the end of experiments (16 hrs), the cells were lysed and collectedfor assessment of intracellular APP levels.

15 μl of medium samples and 15 μg of total protein from each lysedsamples were loaded onto 10% trys-glycine gels, and the protein wasseparated at 150 V for 90 min. The gels were transferred ontopolyvinylidene difluoride paper and probed with an affinity-purifiedanti-APP antibody (22C11), which recognizes the ectodomain of APP(residues 66-81). The APP signals were detected by chemiluminescence andexposed to film. The quantitation of the signals was determined by usinga CD camera and NIH-IMAGE (version 4.0).

Phenserine significantly decreased steady state levels of extra- andintracellular βAPP in dose and time dependent manner (FIGS. 12A and B).Under these conditions, there was no significant toxicity as assessed byan LDH assay.

Screening for Inhibitors of APP

1. Synthesis of Compounds of Carbamate Non-Carbamate Compounds

N,N-Dimethyl-N1-benzyl-5-methoxytryptamine (MES 9191)

N,N-Dimethyl-5-methoxytryptamine (654 mg, 3.0 mmol) and NaNH₂ (234 mg,60 mmol) were added into THF (10 ml), then benzyl bromide (513 mg, 3.0mmol) was added. The mixture was refluxed under nitrogen with stirringfor 2 days. Workup gave MES 9191 281 mg (30%).

(−)-(5aS)-3,5a,10-Trimethyl-1,3,4,5,5a,10a,10-heptahydro-1,3-diaza-2-one-7-hydroxycyclohept[2,3-b]indole(MES 9205)

Method 1:¹⁵ Eseroline (180 mg, 0.82 mmol) and trimethylsilyl isocyanate(48 mg, 0.42 mmol) were dissolved in toluene (2 ml) in a sealed tube.The reaction mixture was stirred by a small magnetic bar and heated inan oil bath for 6 hours. The temperature of the oil was maintainedbetween 100 and 110° C. After cooling to room temperature, theprecipitated crystals were filtered and recrystallized from MeOH to givecarbamate MES 9205 (64 mg, 30%): m.p. 174-176° C.; [a]_(D) ²⁰=−154°(c=0.5, EtOH); CI-MS (NH₃) m/z, 262 (MH⁺); ¹HNMR (CD₃OD): 6.59 (d, J=3.0Hz, 1H, C6-H), 6.53 (m, 1H, C8-H), 6.43 (d, J=8.0 Hz, 1H, C9-H), 3.88(s, 1H, C10a-H), 3.20 (m, 1H, C4-H′), 2.95 (m, 1H, C4-H″), 2.84 (s, 3H,N10-CH₃), 2.62 (s, 3H, N3-CH₃) 2.19 (m, 1H, C5-H′), 1.81 (m, 1H, C5-H″),1.30 (s, 3H, C5a-CH₃). Anal (C₁₄H₁₉N₃O₂) C, H, N.

3-Methoxy-(1′-N-methylamino)-ethyl-benzene (MES 9271)

3-Methoxyacetophone (2 g, 0.013 mol) with methylamine (6 g) was refluxedin ethanol for 4 h. After evaporation of solvent chromatography gaveschiff base (1.7 g, 80%). The schiff base of 3-methoxyacetophone (1.25g, 0.008 mol) was dissolved in methanol (25 ml) and reduced bysodiuimborohydride (0.24 g, 0.008 mol). Workup gave MES 9271 1 g (80%).

Propionanilde (MES 9291)

N-Methylphenetidine (340 g, 1.85 mol) was dissolved into 750 ml ofbenzene and cooled to 10° C., then α-bromopropionylbromide (200 g, 0.926mol) was rapidly added. The mixture was stirred for 1.5 h at 40° C. thenwashed by water and 1.5%. HCl. Evaporation gave product MES 9291 477 g(100%).1,3-Dimethyl-5-hydroxyoxindole (MES 9292). The propionanilde (MES 9291)(477 g, 1.85 mol) was mixed with 450 g AlCl₃ then heated by an oil bathto 190° C. for 1 h. After reaction the mixture was poured into icewater, the precipitate was filtered and crystallized to give product MES9292 148 g (91%).(+)—O-Methyleseroline (MES 9295). The 1,3-dimethyl-5-hydroxyindole (MES9292) was chiral alkylated by chloroacetonitrile in using the chiralcatalyst CPTC to afford optical rich product,(3R)-3-cyanomethyl-5-methoxyoxindole, which then purified by chiralchromatography to give optical pure(3R)-3-cyanomethyl-5-methoxyoxindole. Reductive cyclization of abovecyano-oxindole by reducing agent red-A1 gave (+)—N1-O-methylnoreserolinewhich then was methylated by formaldehyde and sodiumborohydride to giveMES 9295 in the yields as described in the scheme.

(−)—O-Tetrahydropyranyl-N-1-noreseroline (MES 9320)

Starting material Nitrile (120 mg, 0.4 mmol) and virtride (0.15 ml, 0.4mmol) were dissolved in toluene. The mixture was stirred under nitrogenat room temperature for 3 h, then 5 ml of 5% NaOH was added. The toluenelayer was separated out and the aqueous layer was extracted with ether(2×5 ml). The combined organic layers were washed with brine, dried oversodium sulfate, evaporated in vacuum to give MES 9320 119 mg (92.5%).

1,3-Dimethyl-3-hydroxy-5-methoxyoxindole (MES 9323)

To a 25 ml flask was added benzene (12 ml) and oxindole (95.5 ml, 0.5mmol, then 2 ml of 50% NaOH solution were added. The mixture afterstanding for 12 h was extracted with ether (3×10 ml). The combinedextracts were washed with brine, and dried over MgSO₄. After evaporationof solvent and chromatography (silica gel, petroleum ether: EtOH=3:1)gave MES 9323 62 mg (50%).

2. ELISA Assay for Identification of APP Inhibitors

An enzyme-linked immunosorbent assay was developed to detect secretedAPP in SHSY5Y cells, a human neuroblastoma cell line. The purpose of thescreen was to discover small molecules that inhibit APP proteinsynthesis in SHSY5Y cells. SHSY5Y cells were plated at 1×10⁵ cells/wellin 100 mL/well of DMEM containing 0.5% heat inactivated FBS in 96 welltissue culture treated plates (Falcon no. 35 3072). 144 MES compoundswere tested at final concentrations of 20 mM, 6.7 mM, and 2.2 mM in 0.1%DMSO. Compounds were added and the plates incubated for 16 hours at 37°C./5% CO₂. Maxisorp plates (Nunc no. 437958) were coated overnight with2 mg/mL capture antibody (Biosource 44-100) diluted in Ca⁺⁺ and Mg⁺⁺free PBS. Biosource 44-100 is a mouse mAb that recognizes aa 1-100 ofhuman APP. Plates were blocked for 30 minutes with 1 mg/mL BSA in Ca⁺⁺and Mg⁺⁺ free PBS. Plates were washed 3× with wash buffer (Ca⁺⁺ and Mg⁺⁺free PBS+0.01% Tween20). 50 mL of supernatants and non-cultured mediumcontrols were transferred from culture plates to ELISA plates. Cultureplates were reserved for toxicity analysis. Supernatants were incubatedfor 4 hours at RT on a plate shaker. After supernatant incubation,plates were washed 3×. Primary antibody (Signet Clone 6E-10 biotin) wasadded at 0.3125 mg/mL and incubated overnight at 4° C. The recognitionsite of 6E-10 is aa 1-17 of Ab. For detection, a 1:3000 dilution ofhorseradish peroxidase conjugated streptavidin (Endogen no. N-100) wasincubated for 30 minutes. Plates were washed 3×. Enzyme activity wasassessed by incubation with 100 mL/well TMB substrate solution (Moss no.TMB-US) for 20 minutes. The reaction was stopped by the addition of 100mL/well 0.18M sulfuric acid. Optical density was read at 450 nm onWallac Victor2 plate reader. During the first incubation, toxicity wasdetermined using MTS assay, Cell Titer96 AQ reagent (Promega no. G5430).The background values from the non-cultured medium control weresubtracted from the sample values and secreted APP levels and toxicitywere expressed as % vehicle control. Of the 144 MES compounds screened,12 inhibited secreted APP as determined by ELISA. The results aredepicted in FIGS. 13A-M.

Using the experimental procedure outlined above, APP secretion andtoxicity studies were performed on several other non-carbamatecompounds, and the results are shown in Tables 3 and 4. The compoundstructures are provided in Table 2 in the application, which areidentified by their MES number.

TABLE 3 APP Secretion by SH-SY-5Y Cells Dose (uM) Dose (uM) Dose (uM)Dose (uM) Dose (uM) Dose (uM) Number NIH # MES 0.08 0.25 0.74 2 6 20 6594xp2 9191 100.7666667 101.6333333 99.86666667 100.74 65.3 64.36 15y-1-15 9199 92.2 93.9 102.95 17 y-1-46 9201 97.25 110.75 124.65 18y-1-48-1 (−) 9202 94.4 107.55 110.05 19 y-1-48-2 (+) 9203 103.1103.866667 108.36667 21 y-1-59 9205 115.1 107.4 114.7 96.7333333 96.922.166667 22 y-1-60 9206 88.2 97.1 96.4 27 y-1-66 9215 94.55 108.1 6.234 y-2-1 9222 99.85 105.55 120.4 37 y-2-13 9225 110.45 109.2 59.75 38y-2-15 9226 116.55 101.95 104.65 39 y-2-16 9227 101.15 99.45 120.5 40y-2-18 9228 97.75 110.45 130.05 41 y-2-19 9229 111.6 111.4 125.1 42y-2-20 9230 101.25 88.8 4.4 43 y-2-24 9231 104.9 133.5 154.2 46 y-2-319234 106.4 96.5 98.8 48 y-2-33 9236 105.8 103.2 101.65 49 y-2-34 9237149.95 158.1 173.3 50 y-2-35 9238 117.6 107.7 109.7 48.566666739.2333333 23.566667 54 y-2-43-2 9242 116.75 116.3 116.1 55 y-2-49 924388.95 95.25 100.55 69 y-2-95 9257 111.2 98.9 99.033333 71 y-2-105 925990.7 101.1 111.1 72 y-2-106 9260 93.95 103.2 101.1 78 y-2-113 9266 94.495.2 94.85 79 y-2-114 9267 97.8 97.43333333 97.43333333 93.68 68.6422.22 82 y-2-130 9270 95.8 95.9 98.15 83 y-2-131 9271 96.6 87.95 92.296.1 89.9 63.825 87 y-3-5 9276 96.15 116.95 133.4 89 y-3-15 9277 98.25109 113.6 90 y-3-17 9278 118.4 116.766667 120.63333 91 y-3-19 9279 97.898.4 97 43.2666667 42.3666667 18.4 104 y-3-58 9291 98.2 99.5 99.7100.133333 96.9 67.8 105 y-3-59 9292 106.15 99.55 100.15 106 y-3-60 929397.8 102.75 94.45 107 y-3-69 9294 105.25 90 111 108 y-3-71 9295 102.695.7 89.35 114 SH-281 9301 108.45 106.1 111.15 115 140xp2 9302 100.35105.4 105.8 118 614xp1 9305 106.2 102.4 102.2 119 552xp3 9306 110.75114.55 124.8 122 piy-1-79-2 9309 100 106.4 100.9 123 129xp3 9310 104.897.85 98.95 124 637xp4 9311 102.45 103.05 102.7 125 piy-1-79-3 9312105.4 101.7 96.65 127 piy-1-79-4 9314 111.25 99.15 109.4 129 piy-1-79-59316 104.15 98.45 102 130 597xp4 9317 99.8666667 112.266667 122.03333131 197xF3 9318 104.05 103.4 108.75 132 504xp2 9319 108.95 114.1 112.8133 338xp2 9320 103.15 91.8 15.2 134 574xp1 9321 97.95 98.1 100.5 136111xp1 9323 98.4 102.3 98.3 137 484xp2 9324 110.3 95.9 99.95 143y-1-78-1 9330 99.6 101.5 110.65 144 y-1-78-2 9331 95.45 102.1 106.7

TABLE 4 APP Toxicity SH-SY-5Y Cells Dose (uM) Dose (uM) Dose (uM) Dose(uM) Dose (uM) Dose (uM) Number NIH # MES 0.08 0.25 0.74 2 6 20 6 594xp29191 104.4666667 103.9 103.7 99.18 92.84 91.1 15 y-1-15 9199 100.5101.65 101.9 17 y-1-46 9201 98.9 98.65 98.25 18 y-1-48-1 (−) 9202 99.1597.85 90.25 19 y-1-48-2 (+) 9203 98.93333333 99.16666667 98.83333333 21y-1-59 9205 108.7 105.8 105.6 97.9 97.73333333 33.33333333 22 y-1-609206 96 91.85 76.3 27 y-1-66 9215 92.3 78.6 12.9 34 y-2-1 9222 99.25100.5 98.9 37 y-2-13 9225 104.6333333 100.1 66.5 38 y-2-15 9226106.3666667 99.7 97.9 39 y-2-16 9227 103.6666667 99.26666667 96.0666666740 y-2-18 9228 102.7666667 97.6 95.4 41 y-2-19 9229 99.9666666796.06666667 91.46666667 42 y-2-20 9230 100.8333333 86.1333333313.83333333 43 y-2-24 9231 95.7 90.73333333 85.86666667 46 y-2-31 9234102.55 99.75 99.3 48 y-2-33 9236 97.6 101 100.05 49 y-2-34 9237 96.5598.35 99.4 50 y-2-35 9238 101.8 103.8 106.4 41.8 40.46666667 31.6333333354 y-2-43-2 9242 94.2 89.8 80.95 55 y-2-49 9243 99.1 98.4 99 69 y-2-959257 100.5333333 98.76666667 100 71 y-2-105 9259 102.65 102.9 98.75 72y-2-106 9260 99.8 97.2 98.95 78 y-2-113 9266 96.55 97.75 94.15 79y-2-114 9267 101.5333333 102 101.9333333 97.98 96.72 15.52 82 y-2-1309270 99.4 101.1 100.4 83 y-2-131 9271 103.55 103.45 103.1 99 99.475 94.187 y-3-5 9276 92.85 93.5 90.65 89 y-3-15 9277 98.25 98.3 91.1 90 y-3-179278 97.63333333 98.4 96.33333333 91 y-3-19 9279 102.9 101.1 100.168.63333333 46.66666667 38.63333333 104 y-3-58 9291 99.1 100.5 97.1102.3666667 99.86666667 99 105 y-3-59 9292 102.85 103.75 104.9 106y-3-60 9293 100.8 99.85 101.95 107 y-3-69 9294 99.9 98.3 100.2 108y-3-71 9295 101.15 100.4 99.85 114 SH-281 9301 99.15 97.35 95.9 115140xp2 9302 100.7 101.2 99.05 118 614xp1 9305 95.8 96.7333333394.83333333 119 552xp3 9306 102.3 101.45 106 122 piy-1-79-2 9309 100.8598.6 101.1 123 129xp3 9310 101.85 99.9 97.7 124 637xp4 9311 101.1 101.2599.55 125 piy-1-79-3 9312 99.95 103.85 106.95 127 piy-1-79-4 9314 100.15100.85 100.6 129 piy-1-79-5 9316 99.15 98.05 98.75 130 597xp4 931797.26666667 97.23333333 93.5 131 197xF3 9318 96.2 96.45 96.05 132 504xp29319 99.8 101.05 102.7 133 338xp2 9320 92 84 28 134 574xp1 9321 99.9101.15 99.45 136 111xp1 9323 97.2 100.1 101.15 137 484xp2 9324 102.15100.4 98.65 143 y-1-78-1 9330 99.05 98.45 97.95 144 y-1-78-2 9331 100.4599.9 98.952. Effect of Phenserine Analogues on Steady State APP Levels

3×10⁶ SK-N-SH cells were plated on 60 mm dishes with DMEM containing 10%FBS. After 36 hours, cells were incubated with low serum medium for 1hour. Thereafter, the medium was replaced with fresh low serum medium(0.5% FBS). To start the experiments, spent medium was removed andreplaced with fresh medium with and without phenserine analogues. Thecells were incubated with and without compounds for 16 hours. At the endof experiments (16 hrs), 200 ml of medium and the cells were lysed andcollected for assessment of intracellular APP levels.

20 ml of medium samples and 15 mg of total protein from each lysedsamples were loaded onto 10% trys-glycine gels, and the protein wasseparated at 150 V for 90 min. The gels were transferred ontopolyvinylidene difluoride paper and probed with an affinity-purifiedanti-APP antibody (22C11), which recognizes the ectodomain of APP(residues 66-81). The APP signals were detected by chemiluminescence.The quantitation of blots was undertaken by using a CD camera andNIH-IMAGE (version 4.0).

Several of the compounds of the invention decreased the extra- andintracellular APP levels. A few compounds, which have no carbamate groupwithin the molecule, did not show significant reductions ofintracellular APP levels, but did reduce extracellular APP levels. Thetreatment with 5 mM of MES9280 showed some toxicity (increase of LDHlevels and morphological change). The results are shown in FIG. 14.

3. Effect of Phenserine Analogues on APP Translation

3×10⁶ SHSY-5Y cells were plated on 60 mm dishes with DMEM containing 10%FBS. After 36 hours, the culture medium was replaced with DMEMcontaining 0.5% FBS. The cells were incubated with low serum medium for1 hour, thereafter, the medium was replaced with fresh low serum mediumwith and without phenserine analogues for 16 hours.

After treatments (16 hrs) with and without the compounds of theinvention, the cells were incubated with methionine and cystine freeDMEM containing 4 mM of glutamine for 1 hr. After treatment withmethionine and cyctine free medium, 1 ml of ³⁵S-labeled DMEM (100uCi/ml) with and without the compounds were added and incubated for 10minutes. Thereafter, the labeled medium were carefully removed and thecells were suspended within lysis buffer containing with proteaseinhibitors and frozen at −80° C. until immunoprecipitation assay.

APP were immunoprecipitated from 200 ug of total protein in each sampleswith polyclonal antibody O443, which recognizes 20 aminoacids sequencesof APP carboxy terminal, and protein A/G resin for overnight at 4 C.Immunoprecipitated APP were eluted from protein A/G resin with 30 ul ofelution buffer (10% beta-mercaptoethanol). The samples were loaded onto10% trys-glycinegels, and the protein were separated at 150 V for 90min. The gels were fixed with fixing buffer and dried at 80 C for 60min. The dried gels were exposed onto Phosphor Screen (PACKARD,Instrument Company, Inc., Meriden, Conn.) for overnight and the APPsignals were quantitated on phosphor image.

The levels of newly synthesized βAPP were normalized by TCA precipitablecounts. Several phenserine analogues significantly decreased βAPPsynthesis without changing TCA precipitable counts (FIG. 15A). Inaddition, there was no change in APP mRNA levels (FIG. 15B).

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1. A method of inhibiting production of amyloid precursor protein in a mammalian subject in need thereof, comprising administering orally to the mammalian subject an effective amount of a pharmaceutical composition comprising (+)-phenserine.
 2. The method of claim 1, wherein the dosage of (+)-phenserine is from about 0.1 mg/kg to about 100 mg/kg of body weight.
 3. The method of claim 2, wherein the dosage of (+)-phenserine is from about 1 mg/kg to about 20 mg/kg of body weight.
 4. The method of claim 1, wherein the pharmaceutical composition is administered daily to the mammalian subject.
 5. The method of claim 4, wherein the pharmaceutical composition is administered once daily to the mammalian subject.
 6. The method of claim 1, wherein the pharmaceutical composition comprises a slow release system.
 7. The method of claim 1, wherein the pharmaceutical composition comprises a sustained release system.
 8. The method of claim 1, wherein the pharmaceutical composition is administered by a polymer based delivery system.
 9. The method of claim 1, wherein the pharmaceutical composition comprises (+)-phenserine tartrate.
 10. The method of claim 1, wherein the mammalian subject is human.
 11. A method of treating dementia in a mammalian subject in need thereof, comprising administering orally to the mammalian subject an effective amount of a pharmaceutical composition comprising (+)-phenserine.
 12. The method of claim 11, wherein the dementia is Alzheimer's Disease.
 13. The method of claim 11, wherein the dosage of (+)-phenserine is from about 0.1 mg/kg to about 100 mg/kg of body weight.
 14. The method of claim 13, wherein the dosage of (+)-phenserine is from about 1 mg/kg to about 20 mg/kg of body weight.
 15. The method of claim 11, wherein the pharmaceutical composition is administered daily to the mammalian subject.
 16. The method of claim 15, wherein the pharmaceutical composition is administered once daily to the mammalian subject.
 17. The method of claim 11, wherein the pharmaceutical composition comprises a slow release system.
 18. The method of claim 11, wherein the pharmaceutical composition comprises a sustained release system.
 19. The method of claim 11, wherein the pharmaceutical composition is administered by a polymer based delivery system.
 20. The method of claim 11, wherein the pharmaceutical composition comprises (+)-phenserine tartrate.
 21. The method of claim 11, wherein the mammalian subject is human. 